Hi,
I am trying to isolate RNA from Chilli matured leaves (Capsicum annuum) with RNAiso plus reagent (TAKARA) method. The 260/280 ratios are in the range of 1.90 to 2.05, and 230/260 are in the range 1.60 to 1.90. RNA concentration is also decent (around 400 ng/ul). But the RNA on agarose gel looks bad. Ct values for reference genes are also varying sample to sample. Anyone has faced similar problem with chilli? It would be great if someone help me to troubleshoot the issue.
Thanks.
You cannot run total RNA on a plain agarose gel and get accurate sizing. The Ribosomal RNAs have too much secondary structure and will not run as 18s and 28s species. You must use a formaldehyde gel and denature the RNAs first to see 18s run at about 2kb and 28s at about 5kb. .
It looks like you are seeing some degradation of your RNAs. The usual advice is: clean your work area & equipment really well with "RNase away", keep your tissue samples FROZEN at -80C, grind them in liquid nitrogen, and don't let them thaw until you add the first buffer in the kit, and always keep the extracted RNA on ice or frozen.
Other advice is to know the maximum capacity of your kit and don't use too much starting material.
It's totally normal to have different Ct values for different biological samples. That's why you include the housekeeping gene as a reference so you can normalize the expression values.
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