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You have several different complement formulations available. How do I know which type to use?
This decision is based on what cells you need to lyse and from what species the cells are from. Typically any type of complement should lyse cells, however we have made different formulations to enhance performance and reduce non-specific toxicity in certain assays. Listed below (and in our catalogue) are some general guidelines:
• Standard Rabbit Complement is mainly used for HLA-ABC tissue typing or cytotoxicity assays using human T cells.
• Low-Tox complement formulations are processed to reduce the toxicity of the complement. They are used for cells that may be sensitive to non-specific lysis.
- Low-Tox-H is mainly for HLA-DR tissue typing (human B cells).
- Low-Tox-M is for cytotoxicity assays using mouse lymphocytes.
- Low-Tox-R is for cytotoxicity assays using rat lymphocytes.
• Baby Rabbit Complement and Baby Rabbit Complement-Tissue Culture Grade are used for cytotoxicity assays which requires the C to have low toxicity while maintaining a relatively high activity (higher than the Low-Tox formats, but lower that the standard rabbit C). Furthermore, the Baby Rabbit Complement-Tissue Culture Grade can be used for assays requiring the C to be sterile, virus and mycoplasma-free, and low in endotoxin.
• Rabbit Complement MA is screened for use in cytotoxicity assays using monoclonal antibodies against human lymphocytes.
• Standard Guinea Pig Complementis mainly used for hemolytic plaque assays and complement fixation assays. The Hemo-Lo format is used for sheep RBC hemolytic assays, and the Low-Tox GPC is mainly for cytotoxicity and plaque assays.
What is special about the Low-Tox formulations?
This type of complement will have low background toxicity (i.e. few cells will be lysed non-specifically).
What dilutions should I normally use?
This varies in each assay, therefore we recommend that you titrate to find your optimal dilution. Generally, a 1:8-1:16 dilution should be suitable (1:2-1:4 should be used for microlymphocytoxicity/tissue typing assays). Additional information may be found on the product data sheet.
What is the shelf life of your complement?
Generally, the expiry is 2 years from date of manufacture for all of our complement formulations.
Can I use expired complement for my assay?
You may be able to use complement that has recently expired, however we cannot guarantee its performance. Should you decide to try it, we recommend using more (i.e. lower dilution) due to possible loss of activity.
Can I re-use my reconstituted complement?
Yes. If the complement is frozen to -70℃ (in single use aliquots) immediately after reconstitution it can be used, however only one freeze / thaw cycle is recommended.
The isotype of my primary antibody will not fix complement. Can I use a secondary antibody that fixes complement to deplete cells labelled with this primary antibody?
This has been tested in the past but with limited success. Therefore, we do not recommend it.
How do I sterilise complement for assays requiring sterile product?
You can sterile filter your complement using a 0.22 µm filter; however you must dilute your complement first to your optimal dilution before filtering. Otherwise, you may lose complement activity because of the filtration process. You can also use the Baby Rabbit Complement-Tissue Culture Grade which is sterile.
Is it better to use frozen or lyophilised complement?
There is no difference in quality. Lyophilised complement should be stored at 20℃, and frozen complement must be stored at 80℃. The lyophilised product will last longer, is more stable, and is easier to ship (dry ice is required to ship frozen complement).
Is there an expiry date for your Lympholyte® products?
We do not put an expiry date on most of our Lympholyte® products as they are stable for years when stored as recommended (i.e. unopened and protected from light). We have performed stability testing on Lympholyte® that is 5 years old, and found it to work very well.
I have Lympholyte® that is more than 5 years old. Is it OK to use?
If it has not been opened and has been stored properly (i.e. in the dark) it should work; however, it is best to use a recently produced batch.
You have several different Lympholyte® formulations. How do I know which one to use?
Which Lympholyte® you would use will depend on the species from which the lymphocytes are being isolated from, and whether they are being isolated from lymphoid organs or blood. We offer Lympholyte® specific for mouse, rat, rabbit and human lymphocytes (Lympholyte®-M, -R, Rabbit and -H, respectively). If you need to isolate lymphocytes from mammalian blood, we would recommend using Lympholyte®-Mammal or Lympholyte®-H. We also offer Lympholyte®-Poly for the isolation for human polymorphonuclear cells. For the isolation of cells whose density falls outside the range of the premade Lympholyte media, we offer Lympholyte®-1.1, a density gradient medium whose density can be set where needed.
Can I use Lympholyte®-M with peripheral blood samples, in addition to cells isolated from mouse tissue?
We recommend using Lympholyte®-Mammal with mouse blood, however you can use Lympholyte®-M as well. You will not recover as many cells as you would using Lympholyte®-Mammal, and there will be more RBC contamination in the interface.
Why do I have to use a serum-free media when using Lympholyte® for cell isolation?
Serum may cause the cells the clump together which causes them to pellet, leading to lower lymphocyte recovery.
Can I isolate granulocytes using Lympholyte®?
Lympholyte®-Poly is designed for isolating human granulocytes. Unfortunately, none of the other Lympholyte® products will do this, and the granulocytes end up in the pellet at the bottom of the tube. By using a RBC lysing buffer (i.e. NH4Cl) and some washing steps, you can purify your granulocytes from the pellet. For more information, please contact our technical services department.
How do your immunocolumns work?
Our columns isolate cells through a negative selection process, using antibodies to bind unwanted cells. All but the desired cells will be bound in the column. Our columns are non-magnetic.
What is the expected cell yield and purity when used properly?
This varies for each column type, however the yield for the regular columns is normally 25-30% (from the initial cell population run over the column) and for the high capacity columns 15-20% (from initial cell population). The purity for most columns is expected to be greater than 85%. Please see individual data sheets for expected numbers.
Do I have to use your Lympholyte® cell separation media when isolating my cells before loading them onto the column?
We do recommend the use of Lympholyte®; however, any method that will isolate the lymphocytes and remove red and dead cells will suffice.
Can I load more cells than what is specified?
It is not recommended. If you load more cells the purity will decrease.
For the Human immunocolumns, can I run the column flow rate faster or slower than 6-8 drops per minute?
It is not recommended as the recovery and yield will be compromised. If run at a faster rate, the purity will decrease and if run at a slower rate, the recovery will decrease.
Will the columns remove macrophages and monocytes in addition to certain lymphocytes?
Yes. There is specific antibody in the cell cocktail to remove macrophages/monocytes. Very few of these cells will be found in the eluent (1-3%).
Can I reuse your immunocolumns?
No. Once the cells are bound you cannot remove them from the column bed.
Can I freeze your immunocolumns to extend the shelf life?
No. These kits cannot be frozen.
What is the shelf life of these kits when stored at 4℃?
The shelf life will be at least 6 months from date of shipment.
What is the difference between your "newly improved" columns and the original columns?
The new column kits contain everything that you need to run the columns. We have added our Lympholyte® cell separation media, PBS buffer and a red cell lysing buffer (use is optional). Also for the mouse and rat lines, the thumb wheels have been replaced with stopcocks to allow better control of the column flow rate. Essentially, no flow rate adjustments are needed for these columns.
MolYsis™ Pathogen DNA Isolation
Why does human DNA need to be removed before PCR assaying of bacteria and fungi?
The human genome contains unspecific binding sites for e.g. pan-bacterial primers. This may give rise to false-positive signals in PCR assays. Moreover, unspecific binding limits the accessibility of primers to the specific sites on the 16S rRNA gene. As a result, the sensitivity of detection is reduced. Last, the removal of human DNA allows the easy processing of larger volumes (up to 10 ml) by a quick mini spin column protocol which in turn increases the sensitivity of detection of bacteria. Altogether, degradation of human/animal DNA reduces the constraints otherwise connected with universal 16S rDNA PCR using whole blood and other primary body fluids.
How is human DNA removed during blood processing?
Samples are treated with a buffer lysing the human/animal cells, while bacteria and fungi are unaffected. The released human DNA is enzymatically degraded and bacterial and fungi cells are sedimented at the end. The protocol is completed within 45 min (6 parallels). Thereafter, any procedure for DNA extraction can be performed to isolate the microbial DNA, including manual and automatic protocols.
Do I have to change my validated DNA purification system?
Not necessarily. Because of its modular nature, MolYsis™ is adaptable to any DNA purification system. In particular, MolYsis™ Basic, MolYsis™ Basic5 and MolYsis™ Basic10 kits are units that are designed to treat 0.2 ml (pediatric), 1 ml and 5-10 ml liquid samples, respectively, before mini isolation of microbial DNA is performed by other kits, including Qiagen, Roche, Promega, Biomerieux and Epicentre. Make sure that ingredients of DNA isolation kits of other manufacturers are DNA-free (reagents, buffers, spin columns, plastics). If you are uncertain, ask your supplier. Automated DNA isolation products can also be combined with MolYsis™ Basic, MolYsis™ Basic5 and MolYsis™ Basic10 (see below).
Can MolYsis™ be used with automated DNA purification systems?
MolYsis™ Basic (kit for removal of human DNA) has been successfully tested in conjunction with liquid handling DNA extraction systems, including Qiagen QiaCube®, Roche Magnapure®, Chemagen Chemagic® MSM I, Biomerieux easyMag®, FujiFilm QuickGene® 810 and PSS Magtration®. Make sure that the reagents used are DNA-free. Ask your supplier.
What kind of consumables are to be used?
Sterile pipette tips with filters are obligate to avoid carryover contamination during DNA preparation. Also, sterile 1.5 and 2.0 ml polypropylene tubes, as long as not supplied with MolYsis™ kits, should be used. Many manufacturers of plastic consumables guarantee absence of human DNA. Only few suppliers test for the absence of bacterial DNA. We have made good experience using Biosphere® pipette tips and plastic vials (Sarstedt, Germany).
What kind of samples can be processed with MolYsis™?
Samples evaluated for kits of the MolYsis™ Basic and MolYsis™ Complete series include:
Human origin: Whole blood, synovial fluid, pleural fluid, cerebrospinal fluid, ascites fluid, pus, broncho-alveolar lavage, nasal wash fluid, urine
Animal origin: Whole blood from mouse, rat, and monkey, chinese hamster ovary cell culture, monkey renal cell culture
Samples evaluated for MolYsis™ Plus:
What is the sample volume that can be processed?
MolYsis™ Basic is for the treatment of 0.2 ml pediatric samples.
MolYsis™ Basic5 can be used for 1 – 5 ml samples.
MolYsis™ Basic10 can be used for 5 – 10 ml samples.
MolYsis™ Complete5, the complete system of human DNA removal and microbial DNA isolation, is available for 1 ml and 5 ml samples.
MolYsis™ Complete10 allows the processing of 5 – 10ml samples.
What is the sensitivity of detection of bacteria and fungi in blood?
Spiking experiments showed that bacteria and fungi can be detected at low titres in whole blood. For instance, with 5 ml whole blood using MolYsis™ Complete5 and an universal 16S PCR assay (Mastermix 16S Complete) or an universal 18S assay the detection limits are (100%; 6 parallel determinations): Staphylococcus aureus, 12 cfu/ml; Escherichia coli, 24 cfu/ml; Candida albicans, 5 cfu/ml.
Can blood cultures be used for isolation of microbial DNA?
MolYsis™ Plus is a kit specially developed for the isolation of microbial DNA from blood cultures. The procedure includes removal of PCR inhibitors and human DNA and the isolation of microbial DNA from 0.2 ml blood culture. In a study it was shown that bacterial growth in blood cultures can be detected by PCR significantly earlier when using MolYsis™ Plus than the microbial detection system (Gebert et al. 2008; see references).
What are the specifications of the instrument, SelectNA™?
SelectNA™ is a contained instrument for the extraction and isolation of DNA from pathogenic bacteria from blood and other body liquids. The instrument serves the sensitive diagnosis of infectious diseases by supplying highly pure bacteria DNA to universal and other PCR assays. SelectNA™ can be used with single samples or in medium size mode with up to 12 parallel extractions. A strong UV source moving around in the interior decontaminates the instrument from cells and DNA thus eliminating a source of low false-positives during assaying. The pathogen DNA isolation pathway includes a manual, low-hands on time procedure for the removal of human DNA (MolYsis) and the automated extraction and purification of pathogen DNA.
What are the clinical samples that can be processed by the SelectNA™?
The system can be used with any body fluid, including whole blood, CSF, BAL, peritoneal, synovial and other aspirates.
How many extractions can be run by the SelectNA™?
The instrument allows to be flexible for the extraction of samples. You can extract 1 to 12 samples with a single run.
What is the volume of sample processed?
The sample volume processed is 1ml. Liquid material with less volume can also be processed.
What is the processing time of pathogen DNA isolation?
The whole process of sample pre-treatment (human DNA removal), extraction and purification lasts approx. 145 min. The total hands-on time needed is 45min and thus accounts only 50% of the manual pathogen DNA isolation procedure, MolYsis Complete5.
What are the components delivered with the 'Blood Pathogen' kit for the SelectNA™ instrument?
The ‘Blood Pathogen’ kit includes all reagents and consumables necessary for extraction and purification of pathogen DNA (except pipette tips). In particular, DNA-free plastic consumables, including sample tubes, pumps for liquid handling and pre-filled cartidges, reagents for sample pre-treatment (human DNA removal), pathogen cell lysis, extraction and purification are delivered with the kit.
How does the DNA isolation by the SelectNA™ work?
After a short, low-hands on manual pre-treatment for the removal of human DNA the concentrated, bacteria-enriched sample is applied to the automat where it is extracted and pathogen DNA is purified by automated liquid handling. The purification of the DNA is magnetic bead-based. At the end, the isolated DNA is eluted in a 100µl volume.
How is the DNA-free state of all reagents and consumables of the kit guaranteed?
All components of the ‘Blood Pathogen’ kit are manufactured under strict quality management and control for the absence of contaminating bacterial DNA. The guaranteed lot-by-lot rate of false positive results is equal to or less than 3%.
Genomic DNA Isolation
What are the yields of genomic DNA from different samples?
- PrestoSpin D Bug – Bacteria 5 to 80 µg (1-5 ml culture), depending on the species
- PrestoSpin D Fungi – Yeasts and fungi 5 to 7 µg/ml (yeast) or per 250 mg mycelium
- PrestoSpin D Blood & Cell – Blood 2 to 4 µg/100 µl
- PrestoSpin D Blood & Cell – Cell culture approx. 4 µg/106 cells
What are the maximum amounts of samples that can be processed?
- PrestoSpin D Bug – Bacteria: 2 x 1010 cells corresp. to 5-6 ml E. coli
- PrestoSpin D Fungi – Yeasts: 1-2 ml (O.D.600nm > 10)
- PrestoSpin D Fungi – Fungi, mycelium: 250 mg wet weight
- PrestoSpin D Blood & Cell – Human blood: 100-200 µl
- PrestoSpin D Blood & Cell – Cell culture: 107 cells
Are highly viscous solutions a problem?
No. With other kits using silica membrane mini spin and gravitational flow anion exchange columns, viscous solutions pose severe problems because of clogging of the matrix. PrestoSpin columns are highly porous and have a constant flow-through also at increased viscosity.
At which points can the DNA isolation procedure be interrupted?
In principle at any point of the protocols. The reason is that Molzym developed buffers that prevent the degradation of nucleic acids even in crude extracts. Also, incubation times are optimised, and longer periods do not reduce the quality and quantity of isolated DNA.
Why is genomic DNA eluted with heated buffer EB?
During the purification process, bound DNA is dehydrated by alcohol-containing washing buffers (WB, 70% ethanol). Rehydration before elution of large genomic DNA molecules is necessary and enhances at increased temperature.
Does elution buffer EB interfere with downstream applications?
Buffer EB, used for elution of nucleic acids from the column, corresponds to buffer TE (1 mM EDTA, 10 mM Tris-HCl). Generally, this buffer does not interfere with downstream applications. However, in cases of a high proportion of template DNA to other components in PCR and sequencing reactions, a 1:4 dilution of buffer EB is recommended.
Can buffers other than EB used for elution of nucleic acids?
Small amounts of complexing agents (EDTA) are needed for optimal yields. Diluted buffer EB (with sterile, deionised water) can be used with negligible loss of yield. Such diluted buffer eluates can be used directly for sequencing and transfection. If water is desired as an eluant, yields are somewhat reduced (by approx. 10-20%).
DNA-Free PCR Reagents
Why is universal 16S rDNA PCR a promissing option of bacteria detection?
For two reasons universal 16S rDNA PCR is a favourable way of bacteria detection in samples. First, the use of highly conserved regions in the 16S rRNA gene allows the amplification of sequences from any bacterial strain. With Mastermix 16S Primer and Mastermix 16S Complete, assays are supplied that use primers binding to the conserved regions of >345 bacterial species, including Gram-positive and Gram-negative pathogens. Second, because of the multicopy status of the 16S rRNA gene and other reasons, the sensitivity of detection of bacterial DNA has been found to be very high (see below).
What is the sensitivity of detection?
The analytical sensitivity of the assay (Mastermix 16S Complete) is ≤200 fg DNA (P. aeruginosa). Expressed as viable counts per assay (25 µl), 1 (S. epidermidis), 4.5 (S. pneumoniae), 7.5 (E. coli), 11 (K. pneumoniae) and 23 (P. aeruginosa) cfu can be detected.
What reagents can bear contaminations by bacterial DNA?
Principally all. In particular, in our experience, the Taq DNA polymerase and primers are mostly prone to DNA contamination due to the manufacturing processes. Molzym has invested great efforts to develop a purification process for the elimination of bacterial DNA from PCR reagents – guaranteed.
How can bacteria be identified when detected by 16S rDNA PCR?
The direct way is sequence analysis. Amplicons are purified in a simple procedure, using a commercial PCR purification kit (e.g. QuickStep™2 PCR Purification kits, EdgeBio). The purified amplicon is then included in a sequencing reaction.
Can MolTaq 16S and Mastermix 16S products be used for Real-Time PCR?
Yes. All DNA-free PCR products of Molzym are fully compatible and optimised for Real-Time PCR, using fluorescent dye or probe detection systems.
Does Molzym manufacture customised mastermixes?
Yes. For further information and inquiries on customised manufactured mastermixes, please contact us.
For what application do I need the primers?
The primers are used in conjunction with Mastermix16S Complete and Mastermix 16S Primer. Both primers bind to conserved sites within the 16S rRNA gene region amplified in the assays. Primer SeqGP is group-specific including, among others, Gram-positive Staphylococcus, Streptococcus. Enterococcus, Listeria, Micrococcus, Bacillus and Lactococcus species. Primer SeqGN includes Gram-negative Pseudomonas, Acinetobacter, Serratia, Enterobacter, Escherichia, Proteus, Klebsiella, Stenotrophomonas, and Legionella species. Using both sequencing primers, mixed infections by bacteria in the two different groups can be analysed.
Do the primers differentiate between Gram-positive and Gram-negative bacteria?
The sequencing primers comprise large Gram-specific groups, but there are some exceptions. As related to clinical applications, the most important organisms covered by primer SeqGP are the Firmicutes (among others, Bacillaceae, Staphylococcaceae, Streptococcaceae, Enterococcaceae) and Actinobacteria (among others, Micrococcaceae). Exceptions comprise few Gamma-Proteobacteria like Edwardsiella spp. Primer SeqGN includes the Gamma Proteobacteria (among others, Pseudomonadaceae, Moraxellaceae, Enterobacteriaceae, Xanthomonadaceae and Sphingomonadaceae. Few Gram-positives are identified, including Clostridium, Corynebacterium, Mycobacterium and Propionibacterium species.
What to do with overlapping sequences?
Overlapping sequences are the result of mixed strains in the extract that could not be resolved by the sequencing primers SeqGP and SeqGN. Typical examples are mixtures of Gram-positive or Gram-negative or strains. In this case we recommend using a special tool for resolving mixed sequences, Isentio RipSeq (www.isentio.com).
Routine PCR Reagents
Can MolTaq be used for hot-start amplification?
Hot start PCR is being used to avoid non-specific reaction products. For this purpose, the enzyme is complexed with Taq-specific antibodies with the effect that Taq activity is repressed until the antibodies are heat-denatured near the optimum of Taq reaction. MolTaq is designed for routine amplification, and here it is superb, comparable in quality to products of competitors. If desired, MolTaq can be used in conjunction with anti-Taq antibodies.
What is the source of MolTaq products?
MolTaq products are manufactured at Molzym’s premises. Each lot of enzyme, buffers and other components of the products are subject to routine quality control.
What is the function of the red dye in MolTaqRed and MolTaqRed Mastermix?
The red dye has two effects: a) one can see what is being pipetted; smallest volumes of Moltaq (0.2-0.5 µl) are visible when being placed in the tube. This reduces the experimental error; b) the solution can be loaded directly after amplification onto the agarose gel (one pipetting step less).
Can MolTaq be used for real time PCR?
Yes, with fluorescent dye and probe detection, respectively.
What is the effect of the PCR enhancer supplied?
The PCR enhancer supplied with MolTaq is used in cases of G+C-rich DNA templates which are often not amplifiable under standard conditions. Reason: G+C-rich DNA tends to melt at temperatures near that of the optimum for amplification (72 °C): PCR primers do not bind properly and, as an effect, PCR products are not formed. The PCR enhancer lowers the melting temperature of the template DNA, primers bind and a PCR product is formed. Another reason for weak or a lack of PCR product formation is internal base pairing of single-stranded template DNA (hairpin structures), which inhibits amplification. The PCR enhancer prevents such structures.
Bacterial Competent Cells
How are cells made competent?
Cells can be made competent either chemically or by electroporation. Chemical competency usually involves treatment with divalent cations at low temperatures, followed by a quick cold-heat transfer during transformation. Electroporation involves the removal of salts that may cause “arching” during the electrical shock. Both methods provide cells that can be frozen for storage.
How do I calculate the transformation efficiency?
(cfu on control plate) / (ng of uncut vector) x (103 ng / µg) x (final dilution) = cfu / µg DNA. (Note: cfu = colony forming units)
QuantideX® qPCR DNA QC Assay
What is the Quantidex DNA assay?
This is a multiplex qPCR assay that measures the absolute copy number of PCR-amplifiable DNA in a sample and reports inhibition to there by guide DNA input downstream. The Quantidex Assay determines the functional quality of sample DNA using the QFI™ Score, which is the fraction of total
genomic DNA copies that can be PCR amplified. The QFI™ Score and copy number of amplifiable DNA provide actionable guidance that can inform the input into NGS target enrichment and help assure analytical sensitivity and specificity. In addition, the Quantidex Assay flags PCR inhibitors in the sample and provides an opportunity to salvage such samples through a subsequent clean‐up step, prior to further processing.
How does the assay work?
This is a multiplex qPCR assay. In the FAM channel the assay targets and detects amplification of an 82bp region in the TATA-Box Binding Protein (TBP) gene in the human genome, which assesses DNA quality & quantity. In the HEX/VIC channel the assay detects amplification efficiency of an exogenous, non-human target that is spiked in to each sample to determine the presence of inhibitors.
How is the QFI™ score calculated?
The assay calculates the absolute copies for each sample via standard curve. QFI™ score is the ratio of actual copies to theoretical copies expressed as a %. As an example, Cell Line DNA which is completely intact should give a QFI™ of 100%. Example: 10 ng of DNA should have 3000 haploid copies (if everything is intact). Let’s say a user added 10ng of DNA in to the Quantidex DNA Assay for a sample, and based on the Ct value, Quantidex DNA Assay determined 300 amplifiable copies. For this sample the QFI score would be 300/3000 = 10% QFI™
How is the presence of inhibitors decided?
The standard curve and NTC should have a certain Ct value for the amplification of the non-human amplicon, because there should be no inhibition in those samples. By comparing the Inhibition Ct value of each sample to these Cts, presence of inhibitors is identified. If a sample contains inhibitors, that will be reflected by either a very high Inhibition Ct or no detection at all for the inhibition component.
What is included in the kit? Do I need to buy any other reagents?
The Quantidex DNA Assay includes qPCR Mastermix, 4 level ready to use Standards, a primer-probe mix for the Inhibition and Quantification portions, ROX and Diluent. No other reagents are needed to perform the Quantidex DNA Assay.
Which Instrument is needed?
The assay should work on any real time PCR instruments that can detect FAM and HEX dyes. It has been validated on ABI 7500, ABI 7900 and Roche LC480.
How long does it take to run the assay?
Hands on time: 15-25 minutes depending on the number of samples. Instrument time: 2 hours on the real time PCR Instrument.
Does this assay replace quantitation by spectrophotometers or Qubit?
It most certainly does. Quantitation by Qubit is not necessary at all. If the user needs to know the QFI™ score, a spectophotometric reading will be needed as that is what determines the theoretical copies of DNA (denominator in the QFI score). But, if a user simply wants absolute copies, then even spec is not needed.
Why is the Quantidex DNA Assay better than Qubit or Spectorphotometry at predicting true DNA quality and downstream success of NGS?
Spectrophotometric methods simply measure bulk DNA concentration without regards to impurity. Qubit simply measures double stranded DNA irrespective of level of fragmentation. For this reason, both of these methods tend to overestimate the DNA concentration, especially in highly fragmented samples types like FFPE and FNA. The Quantidex DNA Assay relies on an amplification based approach to measure the DNA that matters: amplifiable DNA. Since the downstream NGS process is also amplification based, the Quantidex DNA Assay is a better predictor of true DNA quality and downstream success of NGS.
Why is the Quantidex DNA Assay better than other qPCR based assays at predicting true DNA quality and downstream success of NGS?
Workflow: The Quantidex DNA Assay is an all inclusive kit that is composed in to as few tubes as possible and has ready-to-use standard curve material so that the user requires less hands on time to set up the assay. The assay also only requires a single amplification per sample, leading to fewer total reactions for a given set of samples. Other assays might require you to buy additional reagents separately, might require you to perform a serial dilution yourself to create standard curve, may require sample dilution prior to inputing in the assay, or require you to do more than one amplification per sample.
Design: The target gene, as well as the amplicon size for the assay, has been carefully chosen. TBP gene is not affected in cancer and as a result the amplification effects that you see from a sample are purely reflective of sample quality and not due to any other biological changes. Other assays also involve amplicons that are either too small or too large to be a meaningful indicator of how well an NGS library will be amplified.
Actionable Results: The output from the Quantidex DNA Assay identifies any PCR inhibition, reveals the DNA quality score (QFI™ Score), and prescribes volume inputs based on sample-specific copy number for detection of a 5% variant. That is a clear and actionable guidance that goes beyond a simple yes/no answer.
Validation: The Quantidex DNA Assay has been assessed with >2000 FFPE and FNA samples, has a peer reviewed publication (Sah et al Genome Medicine 2013) and is the only assay that has shown a relationship between the upfront DNA QC and downstream NGS variant calls.
How do I analyse the data produced by the Quantidex DNA Assay?
The initial analysis is done on the qPCR instrument as per the instrument’s protocol to obtain Ct values for both the inhibition and quantification portion of the assay. This raw data then needs to be imported to a web tool provided by Asuragen for further analysis. This further analysis will indicate if the sample has inhibitor present. This will also output copies and QFI™ score and guide the amount input needed to attain certain level of mutation calls.
You are amplifying an 82 bp amplicon. My average library size is x bp. How is your QFI™ score applicable to my library size?
Intact DNA samples and other non‐FFPE DNA should be evaluated with the Analysis Software using the assay amplicon size of 82 bp. FFPE DNA can be analysed by using this same amplicon length, or by using an amplicon size equivalent to the median amplicon length produced by your downstream targeted NGS enrichment step and library. In the latter situation, the data analysis software enlists an empirically‐validated algorithm to automatically adjust the amplifiable copy number and QFI™ Score to match the user‐provided library size. This conversion is made possible by the highly predictable effects of PCR‐inhibiting DNA modifications in FFPE samples when informed by the QFI™ Score.