The best way to make sure your cDNA is of good quality is really to check your input RNA. After you isolate RNA in an RNase-free environment (use RNase zap on your pipettes, make sure your water is clean, and always open your test tubes with two hands rather than one when you can), you should....

1. Spec your RNA. The 260/230 ratio should be ideally above 1.8, your 260/280 ratio should be ideally above 2.0, and the peak at 260 nm should distinct.

2. Run an agarose gel of your RNA. You should see two very clear bands indicating that the small and large rRNA molecules are of high quality. If there is smearing of these bands, then your RNA has degraded.

3. If you or your department as a bioanalzyer, use it. It will use microfluidics to give you an RNA integrity number or RIN that will represent how good your RNA quality is. Anything with a RIN above 9 is usually good quality RNA.

If your RNA passes the quality inspections that I've laid out, then you can confidently create cDNA. It's rare that issues with cDNA arise from the cDNA synthesis (reverse-transcriptase in my experience usually doesn't go bad and the buffer is pretty much salts + dNTPs).

I suspect that the reason you only get your band of interest in some samples but not others is that the RNA in the other samples is degraded.

Yousuf Khan Thanks for your answer. Anyway, I am already checked my RNA quality before RT reaction, I got 2 clear band in agarose electrophoresis and good purity with Nanodrop [260/280: 2.133 and 260/230: 1,185]. But there's still no band after general PCR using my actin primer.

Thus, recently, I did qPCR to validate either my cDNA is properly synthesize or not. And surprisingly I got amplification plot and band after running it in agarose.

So how I interpret this result?