Have you check RNA quality in the gel? it may be possible that even though you got good reading in nano-drop RNA may be degraded. A good quality of RNA contain two distinct rRNA band in gel. And also have you check cDNA for pr amplification using house keeping gene.

Have you check RNA quality in the gel? it may be possible that even though you got good reading in nano-drop RNA may be degraded. A good quality of RNA contain two distinct rRNA band in gel. And also have you check cDNA for pr amplification using house keeping gene.

I didn't check for the RNA quality because I don't have electrophoresis in my lab. I checked my cDNA amplification for 1:10 dilution (not a serial dilution) for my house keeping gene and I didn't get any amplification. 

 in my limited experience, you need to do the gel electrophoresis for the rna and cdna to make sure the integrity of the rna and cdna. or maybe you can use fragment analyzer such as Qiaxcel if you lab have it.

A good RNA prep means your 260/230 as well as 260/280 are greater than 2. At least 260/230 should be so, may be 2.2.... It seems your prep is not pure becoz of 260/230 being 1.9. If you cant test RNA quality by electrophoresis then you can just run a regular PCR of GPDH or actin. If you get amplification of correct size proceed to qPCR.  Also check how much (mg) tissue you are starting with. The Qiagen kit does not give good yield if you use more than 30mg tissue. Also you can modify the qiagen protocol by doing trizol-chloroform-isoproanol extraction and then purify the RNA with qiagen column purification. This method gives much higher yields. 

I purify RNA from testis by this method and I get yields up to 3 micrograms. Also I use 2 micrograms of RNA for cDNA synthesis by Superscript III Reverse Transcriptase from Invitrogen. It works best.

Good luck.

• Given that your total RNA quality indices are good; the amount of total RNA you are adding to your RT reaction (1ug) is optimal and also the fact that 2.5mM MgCl(2) is ideal for your RT reaction, on face value this points towards problems with your PCR rather than your RT reaction; Thus, consider the following:
• To completely rule our issues with your cDNA I would firstly validate my cDNA by carrying out RT PCR with a high copy number housekeeping gene such as b actin, tubulin or gapdH: That way if you obtain amplification with those primers/genes and not your GOI then you can infer the problem relates to amplification of GOI which in turn could be primer design; secondary structure or nucleotide bias in your target; the positioning of your primers; and copy number of your GOI. To elaborate:
• Consider how you design your primers. In particular:

• Also, the position of your primers can impact on the efficiency of amplification: Specifically, primers situated within 1kb of your 3' gene terminus (poly A tail) are more efficiently amplified as replication of of RNA proceeds from the 3' terminus so those regions are more accurately and more efficiently replicated. Furthermore, RNA degradation proceeds from the 5' end and so RT is more efficient from the 3' end of the gene, leading to higher copy number in terms of CDNA from those regions and stronger PCR; especially if your RNA is degraded
• Think about the copy number of your gene: Low copy number genes cannot not always be detected by gel based RT PCR but cab be detected (albeit with Ct values > 30 cycles) by real time qPCR (which is on average ~ 10 x more sensitive than agarose gel based RT PCR)
• Keep your amplicons short: If you amplify regions that are 3' biased also try and ensure your amplicons are optimally ~ 100bp for real time qPCR and 100-300 bp foe gel based RT PCR
•  Think about possible secondary in your region of target (as well as primers) you choose to situate your 3' biased primers: High (> 70%) GC content undermines efficient amplification and could result in failure to amplify. Check putative secondary structure in your target using a program like m fold:
• http://unafold.rna.albany.edu/?q=mfold
• If your region is > 60% GC and it is unavoidable designing primers to an alternative region, add 5% DMSO or 1M betaine to both your RT reaction and especially your PCR reaction; increase your PCR cycle number by x 5 and with no amplification try 35-40 cycles (no more !) instead of the usual 25-30 cycles. See:
• http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011024
• Finally avoid target regions with runs of more than 4 x G/C residues (in otherwise normal GC content loci) and AT rich target regions as both of these predicaments will lead to inefficient replication of target during PCR and failure to obtain amplicon bands
• If you are dealing with low copy number targets and inherently difficult target regions and are performing gel based RT PCR as opposed to real time qPCR with your cDNA a high processive polymerase like phusion will always perform better - and give you more amplification product - than Taq

Thank you so much laurance! Really appreciate for all the details. Is there any way I could check my primers if they are good are no? before I go ahead and make new ones. Because the primers I have now, I used most of the guidelines that you mentioned. 

Again thanks for all your help!!

If you can wait a few days I'll post my protocols that I used to design and verify primers (essentially what Laurence already stated) but also extraction/purification/processing DNA and RNA from fresh or frozen tissue (muscle, adipose, liver, pancreas) using Qiagen kits

Dear Sana

take a look at the primer design  document I have attached to my previous answer

In essence, it makes ref to a freeware package called 'Oligo design tool' by IDT and another package called oligo calculator, viz.

• https://www.idtdna.com/calc/analyzer
• Using this package you can screen for heterodimers (primer dimers) and accurately assess Tm
• For self annealing, i.e. homo dimers or hair pin loop, the above IDT package will do the job but personally I prefer the following which is kore straight forward:
• http://biotools.nubic.northwestern.edu/OligoCalc.html
• Before thinking about primer design however, I would consider validating your cDNA with a housekeeping gene if you havn't already done so
• If you have and for example you obtain an amplicon from something like b actin or gapdh then that does indeed suggest primer issues, i.e. design; where they are situated (do you bias them towards the last 1kb of your transcript); amplicon size (is it < 300bp) and copy number of your GOI 

Michael, I would love to look over that protocol. Please send it my way whenever you can.

Laurance, Thanks for all the indepth suggestions. I ran the qPCR today using different dilution series with my housing keeping gene and I didn't get any amplification :( I am thinking to check back my primers or order new ones. I can't think of anything else that I could change besides checking my reagents if they are bad, Although, I bought the kits past summer. 

Dear Sana

• Given that your RNA appears to be of high quality
• Given also that you cannot amplify from 2 independent sets of primers one of which is to a high copy number housekeeping gene by suspicions would fall on the cDNA sample and in particular whether one of your RT reagents, like RT has gone off
• If this were me I would take 1ug of your total RNA and reverse transcribe with fresh RT reagents
• Regarding magnesium check that your 2.5mM MgCl(2) isn't already added to your buffer. If it is and the 2.5mM you quoted is actually the fig they give you for Mg already in the buffer (sometimes they will give you extra mg to increase the conc up to 3mM) then the resultant 5mM conditions might not work
• Alternatively and on the subject of denatured RT reagents if your buffer contains dNTP's and these have been freeze thawed from as common stock too many times dNTPs can 'go off' (they literally become dNDP by losing phosphate) then that would also lead to failed PCR

What ever the specifics the problem you describe does suggest that something is awry with your RT reaction. Do you have an alternative cDNA sample that has worked in the past you could use to test both sets of primers ?

Hi..

Some kits were provide RT-PCR in one Step or Two Step