Hi all,
Hoping for some troubleshooting/advice. Currently setting up a virus typing assay for routine use. I have the assay working with good sensitivity (regularly amplifies samples that are above 30 Ct in detection assays) using Superscript III/Platinum Taq One Step RT PCR (https://www.thermofisher.com/order/catalog/product/12574035) using the reaction in the in the photo and following conditions: RT 50 deg 30 min, 95 deg 15 min, 40 cycles of 95 deg 30 s, 50 deg 30 sec, 72 deg 30 sec, then 72 deg for 10 min. prior to starting T, i relax RNA by 65 deg for 5 min hen cool on ice (with reverse primer in mix), before adding enzyme. Primers are gene specific.
However, majority of our samples are converted to cDNA upon arrival to facility (using random primers), and so it would be easier to adapt this to a PCR assay. On a cDNA plate, I tried the reaction in picture 2 using the same cycling conditions as previously described but obliviously without the RNA relaxation or RT step (starting from denaturation). This was totally negative.
To troubleshoot, I ran a known positive using the OneStep assay as described above in duplicate, however, I stopped the reaction after RT and for the second replicate I took 5 ul and added to PCR as described in 2nd paragraph, whilst leaving the first replicate untouched, and continued the reaction to the PCR. In this case, the 1st replicate (One Step - untouched) worked perfectly whilst the sample split into a PCR didnt work at all (completely negative). This indicated an issue with the PCR itself since the RT must be working fine.
I tried to bring the reaction down to 25 ul with a 3 ul input to see if this would help however this was also negative.
Any ideas or help would be greatly appreciated.
Thanks!
@Jordan hi. Seems by your Last experiment that something from the one step May be excerting inhibition at your platinum TAQ second step.
A)
How about spike the succesful One step reaction to your second PCR? But be aware that the dilution May show up in the agarosa gel.
B) checking for inhibitors of the one step to the second PCR. Prepare first máster Mix (the one step) with out sample. Then do the second PCR positive and negative controles. As positive control use DNA from succesful tubes.
C)
Check your DNA amplification PCR and máster Mix with Just DNA. check temperature protocol fur the just DNA amplification.
D)
No cDNA was produce so no template added. Your previous experiment rules out this, how ever should be concidered no succesful transcription or RNA degradation. then agai your las experimet showed that Reverse transcription was ok. and something may be inhibiting your PCR amp. Back in the 90s, while doing geminivirus in tomatos, run accross the case where samples from visible symptomatic plants where negative, I went and ades 2 microliter up to 5 microliters of sample,,,nothing i showed to my supervisor, he grab his head an told me, i shall dilute sample, so i did 1 in 2; 1 in 10, 1 in a 100 still nothing..shared the results and he kindly said,,, no Luis whwn we do dilution in PCR we do 1 in 1000, upt to 1 in 10 million serial dilutions... is said What??? but I did he was right about in that particular case 1 in 100,000 dilution worked. such is the nature of DNA amplification and inhibitors.
one Question RNAase out is supose to help in preserving sample while in RNA form https://www.neb.com/en/faqs/2013/01/23/is-rnaseh-treatment-required-before-pcr-amplification1 how ever in your case again the discribed experiment tells us all is good in reverse transcription
One question if your one step protocol is working why do you want to change to a 2 step protocol?
Good luck... Hopping you succed!!!
Best regards
Luis
@jordan.this may be useful https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1298932/
I have more question please send cDNA protocol the reverse transcription, isuppose you are not using the one step Reverse Transcription reagents, rather a regular reverse transcriptase?
Sample are purified RNA? What are OD values, DNA was taken of by dnnase?
Best regards
Luis
The Super Script III enzyme you use is designed to perform the RT process in one-step. If you want to implement a two-step RT system to obtain cDNA, you need to use an enzyme that allows you to obtain the first strand with random primers and use a Taq polymerase enzyme in the second PCR.
In my experience in viral diagnostics, we use the enzyme Super Script III, to reduce the time needed to do the two-step RT-PCR, with the indicated primers there would be no necessity to do a two-step experiment, remember that it is sensitive to contamination.
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