Hi Gil,

since you tested really a lot, one question: have you tried ordering the primers from a different company (I had some issues, that were solved this way)?

greetings

I've been ordering from this company for many years, they are the largest primer producer in my country and I and trust them.

But again, I also examined primers that I had previously tested (within the last 6-12 months), in the same lab with same equipment and reagent. They worked absolutely fine at the time and now they don't...

Hi Gil,

Regarding to your "old" primers, already in the lab for some time, they could easily be affected by repeated freeze-thaw cycles, which would produce bad results.

For the "new" literature primers, it depends on how much information they gave you in the paper; sometimes I find that lots of information is lacking regarding the efficiency of the primers, or even the optimal concentration of primers or MgCl2.

You could also have impurities in your samples, which could potentially affect RT  and PCR. In RT, I usually use a mix of oligo dT and random hexamers, which always works well. In PCR, you can try adding BSA in case you have impurities.

Hope it helps

Regarding freeze thaw cycles, these new primers I have been using as 10mM working solutions that I prepared from 100mM stocks. The stocks have only gone through 2-3 defrost cycles, which has never caused degradation in my primers to a detectable scale- this is very concentrated and well maintained DNA. Additionally, it's unlikely that all my primer stocks get degraded at the rate rate.

Regarding new primers, they are drived from several papers in high end papers and degisgned for RT-PCR, where tweaking that magnesium concentrations is not an option like standard PCR.

Mainly, it is highly unlikely that ALL my primers, the new ones and the old ones, suddenly fail - and all are ineffective to the same extant (all have a slope of about 4.0-4.2). this seems much more like an issue with the PCR itself.

As I noted abovee, changed my samples several times, using RNA produced from kits or Trizol, and changed my RT kit (one that also uses a randon hex + polyA mix), so the sample and the RT kit do not seem to be the issue. 

Addition of BSA is not possible with a SYBR green ready mix. But at any rate these are not reactions that I'm trying to get to work for the first time- they worked well and than stopped.

As I see, you controlled for sampling by using different samples sources. You should have controlled the RNA extraction by measuring the integrity of your RNA, because even if the conc and 260:280 ratio of the RNA are reasonable, fragmented RNA sometimes give low efficiency. Do you have No Reverse Transcriptase control to test for RT step? now away from samples and technique, did you change any of the tubes that you were using, some polymers absorb NA to their surfaces, Other inhibitors e.g. Phenol need to be checked for especially you used TRIzol. Presence of Polysaccharides also inhibits the reactions. When you use RNA isolation kits make sure that the membrane is dry from ethanol as it inhibits RT reaction. I hope you can solve it. are your NTC wells amplify?

I used RNAs with measured good integrity.

As I do get results (PCRs don't fail, just work less well) this indicates that the RT did work, even if not at full efficiency. This means I have cDNA that is being amplified- and whatever it is that is lowering PCR efficiency is not due to cDNA.

I did changed tubes twice, and in all cases they were nuclease free low absorbency tubes. 

I used RNA purification columns for the most part, Trizol just once for comparison- same low efficiency results. And I perform the kit protocol as instructed, with ethanol drying step.

my NTC wells do not amplify.

Drying the membrane step is different between different protocols. Some don't ask you to open the tube lid and time is different between 1 min and 5 minutes. you should try opening the lid and centrifuge for 5 minutes.

Hi, I had never get this issue, but you can try with:

- Synthetic RNA or DNA ( are you sure that your problema is the PCR none  RT?, you must check which is the troubleshooting step)

- had you check the quality and purity of you RNA?

- Did you try with another kit of RT?

- Were yours Ct  value low?

regards

Hey Angelica I checked all the things you suggested before and the problem is definitely not there. 

However in the meantime I may have reached a conclusion, it might be the PCR plates. We are using the exact same product we always have (same catalogue number from ABI)  but this seems to be an older batch that might not be adequate for the new machines.

thank, long since moved on form that problem but this is cute and useful :)