Do you transform the RNA in cDNA immediately? Which is the condition to storage your cDNA and RNA?

Do you run a positive control?

Dear Ella

• Having looked at both plots I would say that your signal to noise is low:
• This accounts for your spurious traces but nevertheless at first glance the data still looks basically sound
• Your amplification I also note starts at > 30 cycles suggesting that the copy number of your genes is low. This in part explains your low signal to noise
• What do the traces from your housekeepers look like ? Do they give Ct values ~ 10-20 cycles
• If these traces include your housekeeping primer amplifications then you have problem with low PCR efficincy for sure
• That could be due to low quality cDNA preps which in turn might be the result of poor quality and/or quantity of RNA. How much RNA are you putting into your RT reactions and what is the 260/280 and 260/230 ratio of your RNA ?:
• Your RNA should have a 260/280 ratio > 1.7 and ideally 1.8_2.1 and with that RNA you should use > 200ng in your RT reaction
• I notice that your second dissociation peaks are are just one or two wells: If this corresponds to a single well for particualr gene(s) and the other duplicate/triplicate technical replicates are Ok then simply exclude them from your final analysis
• Extra lower melting curve peaks can correspond to primer dimer (which relates to primer design) but unless you see this extra peak consistently with a particualr primer and if you have 2-3 other techncial replicates with a single melt curve for that primer I wouldn't worry too much (just exclude these outliers from the final analysis)
• I would finish by saying that because occasionally you obtain spurious data from the odd well make sure you have an N of 3/4 for techncial replicates
• I would also say that if you see additional spurious peaks in particualr wells as a consistent well related phenomenon I would clean that well or recalibrate your machine
• If the effect is consistently related to a particualr primer pair then stop using those primer(s) and redesign, taking into account algorithms that deliberately screen for prier (homo or hetero) dimers:
• To that end find a primer design SOP of mine which talks about how you do that

Actually I tested a primer prior to this run and it was all fine then I tested other primer which is this one for the same samples at the same day but this is what I got . I converted my mRNA into cDNA after storage of my isolated RNA in DEPC free water at -80c for less 2 days

I should note that I have diluted my samples (cDNA) by 10 folds

Dear Ella Hasan

I think you must recheck your primers, simply by matching with gene sequence from ncbi gene bank,

be sure about purity of isolated RNA ,

and conc of RNA used to convert to cDNA

sometimes the RT-PCR machine itself need calibration and optimization

Best wishes

Assuming:

I'd say that the problem is you don't have enough cDNA template in your reactions.

You problem seems to be:

a) from the melting curves you seem to have a product (1 peak at one Tm and probaby with different starting concentrations) in a number of reactions, many are negative and some have by products

b) you see no amplicifations curves

At the first sight you seem to have a big baseline problem. As you can produce melting curves I think you use an intercalating dye.

Can you analyse the curves with a melt peak apart form the other reactions? Please do that and show me the curves without and with baseline setting and represented at a log-linear Y-axis.

Dear Ella...

I think it's highly related with the low signal which in turn results from either the lackness of targeted sequence or primer failure!

Regards.