I have RNA , yield concentration 149 ng/ul in 50 ul stock. Now I want to synthesis cDNA using Quantitech Reverse Transcription kit (Qiagen). In the Protocol they suggest maximum 1ug of RNA to use in a reaction. I will use normal end point PCR for my samples. if my stock concentration is ok then how can calculate 1ug RNA from the stock?
You do know that 1 ug is about 6.7 ul of your stock. If that is too much volume, I would precipitate the RNA with ethanol (5 ul Na Acetate pH 5 plus 125 ul ethnaol) pellet the RNA and resuspend it in a smaller volume.
You do know that 1 ug is about 6.7 ul of your stock. If that is too much volume, I would precipitate the RNA with ethanol (5 ul Na Acetate pH 5 plus 125 ul ethnaol) pellet the RNA and resuspend it in a smaller volume.
You are plenty of RNA. Your problem is that it is too diluted. Try to concentrate as Gregory says
If you´re using Quantitect Reverse Transcription from Qiagen, that concentration is ok. Before the RT reaction, you perform first a genomic DNA digestion with gDNA wipeout buffer. That reaction is made in 14 uL: 2 uL of buffer 7x, 6.7 uL of your RNA sample (= 1 ug) and H2O to a final volume of 14 uL. Then it´s the RT reaction in a volume of 20 uL. You don´t need to concentrate the RNA, it´s ready to start working with it. But make sure first its quality is good enough.
Thank you very much guys. Now i understand. Actually I was confused about the final volume of the reaction if i take 6.7 ul of RNA then how much would be the water. because I thought volume of water and RNA solution should be same. Thanks to Sandoval for your clear answer.
here we use 2micro gram RNA for a 20 micro liter reaction... if your RNA yield is low(RNA diluted) you could take more template... but keep in mind one thing that your RNA is pure its A260/280 ratio is more tahn 1.8 and it woult be best if A260/A280 is more than 2.0.
Ya, the 260/280 ration of my RNA is arounf 2.00-2.08. But few have 260/230 value less than 1.
Remember that 260/280 ratio only tells you about purity, but not quality. So, your RNA could be degraded but still show a good 260/280 ratio. The easiest way is to check the RNA (500 ng is enough) on denaturing agarose gel (even normal agarose gel works fine for this purpose).
With this concentration you can still work, but checking the quality of your RNA on the denaturing gel would be a good quality check and further you would get to know the ratio of 28S and 18S RNA. Only after gel analysis you can proceed for cDNA conversion.
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