what is the 260/230 ratio of your RNA? you might have organic carryover that is inhibiting qPCR, you want this ratio ideally over 1.5, the higher the better.

Adding more DNA template to qPCR will sponge up the primers and could result in higher Ct values. How much are you inputting per reaction? or inputting into cDNA synthesis? I recently did a miR Taq cDNA synthesis and the recommended input was 10ng. I called to technical service to confirm since it seemed way too low.

Keep in mind that having a tissue lysate will be different from a cellular one and the housekeeping genes you use for cellular analysis might not be good for tissue. maybe try an rRNA gene like RPL13a or RPL0?

• Very good answer Julie
• To add to what Julie said, perhaps look up papers which use Thymus and see what housekeepers are recommended
• Others so exist such as b actin, B-2 Micro globulin 18srRNA etc
• It is important though to still ensure that your 260/230 ratio is not much less than 1.0 as this implies salt contamination in your starting RNA - particularly GITC from lysis buffer and that could inhibit RT culminating in low RT efficiency
• You should also perform a serial dilution of a pooled RNA sample and make sure your primer efficiencies are > 95%:
• If not that could mean poor RT conversion, especially in your 260/230 ratio 200ng of total RNA: If much less than that even with these more sensitive kits, in my experience, that can culminate in low level cDNA
• Also when you perform RT, forget the new advice about 15 minute reactions: it is always better to do 60-90 minute reactions

Hi,

have you ever run your RNA on Agarose gel to make sure you have qualified RNA for cDNA synthesis?

some times results of nano drop shows great amount of RNA but they are actually degraded RNA

i suppose your 3 days culture may caused RNA degradation

Julie Ann Dougherty,

Thank you for your input. My 260/230 ratios were a bit lower the 1.5, so maybe the next best step would be to do another clean up? What would you recommend for the clean up?

To answer your second question, I put 4 uL of cDNA at a concentration of 1 ng/uL. The total reaction volume is 10 uL.

Laurence Stuart Dawkins-Hall,

Thank you for your input! I think you guys are correct with the 260/230 ratio, so I will perform an additional clean up. For the RT, do you have a protocol for the 60 - 90 minute reactions that you would recommend? I've always just used the iScript protocol which gives you your cDNA after about 20 minutes.

Leila Abtahi,

I had not considered this. I had only ran my qPCR product to see if anything was being amplified. This would definitely be something to try. Thank you for your time!