I'm studying on 5 miRNAs under chilling stress in tomato plants. I would like to know the best way for the calculation of required total RNA for cDNA synthesis. I have obtained the concentration of each sample using a Nanodrop spectrophotometer in ng/ul. According to the cDNA kit guide we need 1ul RNA template for 20 ul total volume. According to the kit guide the procedure could be used for 1 pg to 1 μg of total RNA or enriched microRNA. I have attached the concentration of my samples and mi kit guide to this question. I would be thankful if you could share your valuable experiences with me. Thank you so much for you time and consideration.
The protocol you attached allows for up to 9 µl of RNA to be used in each reaction. If you want to make cDNA from 1 µg of RNA then you should add the required amount for each reaction and bring up to a 20 µl final volume with water. If you want to use less RNA for each reaction (like 100 ng), then you will need to dilute the RNA samples down.
Maybe. The only way to know is to test a few samples and see if the Ct values are inside the linear range of your standard curve.
I'd recommend doing just a few samples at a time and then scaling up.
Good luck!
Dear Yasamin
At first, because I am working on plants too, I recommend considering you can get much more higher concentration of total RNA, which can be helpful in cDNA preparation because a higher concentration means lower amount of RNA in cDNA preparation.
secondly, in cDNA preparation: I do the same as Marta 1/0.284
follow the below link
Article Impact of RNA degradation on gene expression profile: Assess...
- Badges
- Science method