Hello,

The kit instructions read that it is optimised for qPCR, but there are so many master mixes in the market that very likely they optimised it for their own qPCR mixes. It produces single-stranded cDNA using poly-dT and random hexamers (so the cDNA covers both the 3' and 5' regions of the mRNA, and due to the random hexamers it reverse transcribes other RNA's as well) of at least up to 12.5 Kb. Also, it cleans the mix from genomic DNA.

In other words, yes, your cDNA should perform fantastic in RT-PCR as well.

To troubleshoot, check these questions:

- does the RT-PCR mix work with other templates/primers?

- do the primers you use amplify other templates? If no, your primers are off or badly designed. If yes, either your cDNA is off or you are using primers that bind to genomic but not to mRNA.

- if the answer above is yes and the primers are correct and bind to mRNA, what steps did you follow to ensure integrity of your RNA? If it is degraded (in this case you better discard it for qPCR or semi-quantitative RT-PCR) and you are trying primers that span a big amplicon (>1 Kb), very likely you won't get much amplification if at all.

Good luck,

Ruben

Hello,

The kit instructions read that it is optimised for qPCR, but there are so many master mixes in the market that very likely they optimised it for their own qPCR mixes. It produces single-stranded cDNA using poly-dT and random hexamers (so the cDNA covers both the 3' and 5' regions of the mRNA, and due to the random hexamers it reverse transcribes other RNA's as well) of at least up to 12.5 Kb. Also, it cleans the mix from genomic DNA.

In other words, yes, your cDNA should perform fantastic in RT-PCR as well.

To troubleshoot, check these questions:

- does the RT-PCR mix work with other templates/primers?

- do the primers you use amplify other templates? If no, your primers are off or badly designed. If yes, either your cDNA is off or you are using primers that bind to genomic but not to mRNA.

- if the answer above is yes and the primers are correct and bind to mRNA, what steps did you follow to ensure integrity of your RNA? If it is degraded (in this case you better discard it for qPCR or semi-quantitative RT-PCR) and you are trying primers that span a big amplicon (>1 Kb), very likely you won't get much amplification if at all.

Good luck,

Ruben

Thank you very much Ruben for your informative discussion, I have checked the RNA using NANO and gel, they are fine both purity and integrity. My primers were taken from a Journal, and you know it is gene specific, how can it amplify other templates?

my 1st primer didn't work, then i thought it might be primer problem. then i changed to another which was also taken from another Journal. but still not result.   

Hello 

Rubens answer is excellent to I will just qualify one or two of his remarks:

1.  To assess the quality of your cDNA also pay attention to the 260:230 ratio as well as the 260/280 ratio. A lot of persons assume that if the latter is 1.8-2.2 then the RNA is of high quality. What it actually means is that protein contaminants have been effectively removed. This RNA might still contain salts and in particular GITC present in lysis solutions and for column based purifications the first wash buffer

2.  This salt is designed to inhibit RNAses and provide a stable extraction environment ( in the case of lysis buffer) and denature proteins on the column ( in the case of the first wash buffer)

3.  If your 260/230 ratio is < 1.0 this indicates that salt - probably GITC - has made it into your final eluted purified RNA and this contamination can inhibit your RT reaction culminating in failed PCR as you have described 

Ideally you need to keep this ratio greater than 1.0 and preferably > 1.5;  one little improvisation that can facilitate this ratio by improving desalting is to allow the ethanol wash buffer to sit on the column for 1 min before spinning through and then repeat this wash

4.  As already suggested try another known set of primers to validate your cDNA; preferably primers to a high copy number universally expressed gene like a housekeeping gene e.g b actin b Tubulin or GAPDH

5.  Conversely try your new primers on a cDNA that has already been validated but remember to include a second reaction with this validated cDNA using a second set of validated primers ( like the above); in effect serving as a positive control

6.  In terms of primer specificity and extra bands these can come about through non specific secondary amplification invariably caused by low stringency reactions due to an annealing temp that is more than 5C lower than the lowest primer Tm

7.  For high stringency specific PCR as a rule you should keep your annealing temp at Tm -2 to Tm - 5C although for new generation engineered highly provessive polymerases like phusion which replicate very efficiently the actual Tm can be utilised as the annealing temp and still yield specific yet substantial that is high efficiency amplification

8.  When calculating your Tm also remember to calculate your Tm using a reputable calculator like IDT oligo analyser which take account of your mg concentration; primer concentration and dNTPS:  these principal parameters affect the stability of your duplex in your local environment and thus your local Tm

9.  Given that that your primers however are taken from papers with validated conditions I suspect the issue has something more to do with initial RNA and derived cDNA ( although occasionally I have encountered problems with primers taken from papers so tend to design my own)

 10. I have commented on the importance of salt contamination and adducing such contamination using the 260/230 ratio and Rubén has made excellent remarks regarding cDNA synthesis so i will stop there 

https://www.idtdna.com/calc/analyzer

Thank you very much Stuart for your long discussion. However, the RNA quality both 260/280 and 260/230 are very good and in the the recommended range. will try with GAPDH and see how.  i have designed a new primer, will try with that. Thanks a lot.

Agreed most likely a primer problem rather than a cDNA quality problem? Do you have standard primers that should work on any cDNA pool (ubiquitously expressed)?

Hello again

it appears there is a consensus building up around there bring a likely problem with primers necessitating redesign

if you are contemplating redesigning primers then PRIMER 3 is a good program or to ensure that your primers cross contiguous exon boundaries and therefore selectively target mRNA PRIMER BLAST ( see below)

Alternatively ROCHE has an excellent primer design tool for intron spanning qPCR primers

i tend to cross ref primers identified in gen bank with primer BLAST against the Roche tool and then further screen for possible primer dimer/ self dimerisation using a couple of free software tools (also) specified in a word doc I prepared which discusses optimal primer design ( and to which I provide a link in my drop box folder).  Enough said !!

see next answer for links: 

https://lifescience.roche.com/webapp/wcs/stores/servlet/CategoryDisplay?catalogId=10001&tab=&identifier=Universal+Probe+Library&langId=-1

https://www.idtdna.com/calc/analyzer

http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi

And finally..,,

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

https://www.dropbox.com/s/m4vfq4wjscnkm7w/PRIMER%20DESIGN%20document_updated%200414.docx?dl=0

Dear Stuart, you have done a great job for me. Thank you very much.

Hi I have taken a copy of your primer design protocol, Laurence, very clear and nice! If I can add something, there is an updated version of primer3 at primer3plus.com/, and I think it's worth mentioning that there are different algorithms for Tm calculation... and Tm's can vary more than 10 degrees celsius between them! So my suggestion would be to stick to the same software and algorithm (I personally use the SantaLucia 1998) and get used to empirical optimisation when required.

I have a short review on primer design among my publications, that I'm happy to share too if anyone is interested.

Excellent discussion, it's great to see so much support!

Best,

Ruben

Hello Ruben

thank you for that. Yes algorithms vary based on predictive model nearest neighbour A/T = 2 G/C = 4 etc and also local conditions which affect duplex stability. I always try and emphasise the need to state things like mg etc for the reasons you have given and I tend to recommend oligo calculator for hair pin loops and IDT for primer Herero dimers based on interface. But yes u very much take your point and will modify my SOP and also look at your site. Very best wishes and thanks for your kind words. Laurence