Hi

It seems your RNA band is quite good. Additionally you can check the quality of your RNA in Nano-drop. If the A 260/280 ratio is good enough you can proceed for cDNA synthesis.

Thanks

Hi,

for qRT-PCR you normally amplify shorter fragments in a range of 100-200bp. Even in case of fragmentaiton this is not as harmful as e.g. for cloning PCRs as the required cDNAs do no need to be full length. So I would agree with Maihuddin and say the samples are good to go for cDNA synthesis

Sven

1) Seems you have some degradation, it should be fine for qPCR, probably not so for RNAseq. You should use comparable RNA quality, for example is not a good idea having intact RNA in the control and quite degraded RNA in your treatment (although I think if you are going for relative quantification, normalization with good reference genes should help to minimize the problem). If your RNA quality is suboptimal, using random examer rather than oligo-dT is usually a better choice. In your case, I think your RNA is still ok (no smearing between the 2 bands of rRNA) but I would prefer random examer.

It is important to understand if you actually got some degradation in your stock, or it occurred during the ELFO. Was your tank, agarose, TAE buffer RNAses free? Did you use any protection against RNAses (formaldehyde in the buffer, sodium hypochloryte of the bleach gel, formamide in the loading dye).

2) Some RNA loading buffer, example from ThermoS, use formamide instead of glycerol, works well to protect your sample and to achieve some denaturation, which means sharper bands as Laurence said. I skip the heating and ice step (which is the protocol as per leaflet instructions) as I usually do not see much of a difference.

4) It is not DNA what you have left in your wells? As per MIQE guidelines you have to proof (with a dedicated control) that you do not have significant genomic DNA contamination. I think this apply regardless if you use a DNAse step or the primer design strategy (spanning the junction etc)

yes