QGE FAQ’s

What is the standard quality and quantity of DNA for iPLEX QGE reactions?

• We recommend an A260/A280 ration of 1.7 – 2.0 and 5 – 10 ng/ul of DNA per reaction well.

What is the maximum plex level per well for iPLEX QGE?

• The maximum plex level for iPLEX QGE is a 29 plex. 

Is extension primer optimization really important for my iPLEX QGE reactions?

• Yes!  As oligo of higher molecular weights fly slower than the lower molecular weight oligos, you need to ensure that your extension primers of are as equal intensity as possible.  The rule of thumb is that your lowest intensity peak is at least 50% of your highest intensity peak.  You can choose to do a 2-step, 3-step, 4-step or linear regression to optimize your primers.  The primer optimization method is located in Appendix A of the iPLEX QGE Application Guide.

How do I design my assays?

• You should have a tab deliminated text file with your gene name in one column and the Ensembl transcript ID in the second column.  With this file you can log into realSNP.com and use the exon extraction tool to extract your exon-exon boundaries of your genes of interest.  You can further put this file into the Assay Designer software either on your desktop or on the realSNP.com website.  The direct instructions are available in the iPLEX QGE Software Guide.

How do I get a My SEQUENOM account?

• Please visit MySEQUENOM.com and register online.

Why should I order my PCR and Extension Oligos from a separate oligo house where I get my Competitor synthesized?

• Most big oligo houses purifies all of their oligos over the same column resulting in the low-level contamination of PCR primers with competitor if the PCR primers are synthesized after the competitor or ordered anytime after the competitor synthesis.  We see contamination up to 3+ months after the competitor synthesis to date.  This issue has become very apparent for our prenatal diagnostics studies.  Primers and competitors should therefore always be ordered from distinct sources if possible.  If not, the current recommendation that the PCR primers be ordered first and tested, followed by the ordering of the competitors, must be followed. PCR primers should not be reordered from that oligo house for a very long time.

Which Oligos do I need to have purified?

• We recommend standard desalting for your PCR and Extension Oligos and either PAGE or HPLC purification for your Competitor Oligos.

Is doing a volume check prior to spotting my plates an important step in the overall Genotyping process?

• Yes!  The chip is what bridges the gap between the reaction plate and the mass spectrometric analysis.  If material is not properly spotted on the chip, you will have bad results.  Make sure your liquid level is the same for all wells (no evaporation problem).  Make sure your pins have been conditioned properly (daily and weekly maintenance).  Check the resin in the well to make sure that it is all at the bottom of the well.  You can find how to perform a volume check in your nanodispenser’s manual.  The volume range for good results is 15-40 nL.  An average volume of 25 nL is a good target to shoot for.  If you observe high pin to pin variability during volume check, clean and condition your pins.

For Housekeeping genes, is there a database available where you can check in which region the genes are expressed in a certain tissue?

• Yes, please refer to the human gene expression index or HuGEIndex for expression patterns based on microarray data: http://132.183.242.155/hio/databases/index.html

What cDNA synthesis kit do you recommend using?

• Internally, we mostly use the ThermoScript™ RT-PCR System for First-Strand cDNA Synthesis from Invitrogen in order to reverse transcribe at higher temperatures than SuperScript allows.  It has been successful for us to date.  

Allele Specific expression and Quantitative Genotyping.  There has been a lot of interest in quantitative genotyping for applications such as looking at gene copy number or LOH in cancer, quantitating the presence of a mutation in either a mixture of normal and tumor cells or in a heterogeneous population of tumor cells, quantitating the number of transgenes present in a cell and in genotyping plants that may by polyploidy.  It sounds like regardless of whether you are analyzing RNA or DNA, if you want to quantitate a nucleic acid in which a SNP or mutation is present you would use the QSNP assay designer.  Is this true?

• There are pretty much three assay design options for quantitative applications:
  1. Standard SNP Designer-covers relative, quantitative genotyping such as allele-specific expression, LOH, allele frequencies in pooled populations, gene-copy number, polyploidy, etc.
  2. QSNP or iQSNP Designer -this designs a false allele against 1 or 2 wild-type alleles in a genomic/copy DNA sequence (meaning a sequence without delineated exon/exon boundaries) to allow for absolute quantification with a competitor.  Its applications are gene copy number or other assays using genomic DNA, allele-specific expression using three alleles, one of which is the competitor allele, etc.  In general I call these Quantitative Genomic Assays (QGA).
  3. QGE Designer-this designs a false allele in a transcript sequence to allow for absolute quantification of cDNA using a competitor template.  Quantitative Gene Expression (QGE).
• The main difference between QSNP and iQSNP and QGE is that QSNP and iQSNP works for genomic templates without delineated exon/exon boundaries but with delineated SNP sites whereas QGE Designer requires exon/exon boundary delineations in the input file but no SNP sites. 

Can you multiplex to the same level with QSNP and iQSNP as you can with iPLEX and still get reliable quantitation? 

• No, 40-plexes cannot give reliable relative peak height data of the quality required for good quantitation.  We recommend not going over 29-plexes as lower plexes show better results.

• When running the assays, are they analyzed using allelotyping or is there a separate application for analyzing quantitative genotyping or allele specific expression?

• Relative quantification assays are run with TYPER Analyzer using Allelotyping.  Absolute quantification assays for gene copy number or allele specific expression, etc. should be run with the QGE Analyzer so that the standard curves can be viewed and the EC50 of each allele can be calculated by the software.

You also mentioned that you’d use double stranded competitor for analyzing DNA.  Synthesizing a long oligo is expensive and I know that a number of customers would like to avoid doing this.  Isn’t possible just to divide you result by a factor of 2?

• The double-stranded competitor acts to ensure that the wild-type template and competitor are amplified with the same kinetics during competitive PCR.  Users can make the competitor double-stranded via PCR if desired and then purify with a column.  Actually determining a bias in competitive PCR is a difficult task since PCR is exponential the templates are competing for the same primers.

Does the QGE software calculate the absolute quantity of for both alleles and also the ratio or is this also a case in which peak areas have to be exported to something like excel to make all the relevant calculations?

• The QGE software does calculate the EC50 for both wild-type alleles in the case of allele-specific expression; the allelotyping software calculates the ratio of the alleles against each other.

Gene copy analysis.  There are potential clients that are interested in doing gene copy number analysis.  In this case there isn’t a SNP involved but they want to know how many copies of a particular gene are present in genome.  Am I correct in assuming this can be done in our system just by running QGE on DNA instead of RNA?  Is it as simple as entering your DNA sequence into the QGE assay designer?  If not what are the differences and are there ways to getting around them in the software?

• Gene copy analysis on genomic DNA is what QSNP or iQSNP designer is for.  A false allele is created at a user specified site and a competitor is created along the PCR and extension primers.  The QGE analyzer is used to calculate the EC50 from the standard curve.