Dear all,
I am a bit confused about the RNA concentration to be used for cDNA Synthesis. I extracted RNA from 3T3-L1 cells. The 260/280 value is 2.21 and 260/230 value is 2.2 which I think is acceptable and good. The concentration is 538.8 ng/microliter in a total 15 microliter volume.
So, 538.8 x 15 = 8.08 micrograms of Total RNA. Is this correct calculation for measuring RNA in micrograms to be used for cDNA synthesis ?????
The 2nd thing is that, in the protocol they say that mix RNA (...μg/Reaction) but there two different volumes.
1. Template RNA + primers+ water = 5 μl which is to be heated at 70 °C before mixing with the
2nd volume
2. The 2nd volume is the mixture of nucleotides, reaction buffer, reverse transcriptase etc = 15
μl. Mix this 15 μl with the 5 μl from step 1 and proceed for annealing and extension.
Now here the amount of RNA to be used (lets say 1 μg/reaction) for cDNA synthesis should be calculated based on the 5 μl volume of step 1 or 20 μl volume (Step 1+ step 2 volume)????
Your reply would be highly appreciated.
Hello Mr.Ahmad, Your RNA concentration is good. For cDNA synthesis you should read the protocol of your cDNA synthesis kit. For example, in the BioFACT cDNA synthesis kit there is a need of about 10ng to 10 microgram of total RNA for example if you need 1 microgram of your total RNA for cDNA synthesis you need 1.9 microliter of your total RNA. Its on you synthesis kit. Good luck.
Yes, as what Ali Khalafizadeh pointed out, you only need to pay attention to the amount you acquire from the total RNA stock, which is 1.9 microlitre. You do not need to judge whether the amount is based on the 5 microlitre or 20 microlitre reaction; the amount you need is in mass and mass is constant. Unless in another scenario that requires you to add amount by concentration, then you need to pay attention to the volume. Essentially, what you need for 1 microgram of total RNA is 1.9 microlitre or 1 microgram/20 microlitre in concentration (20 microlitre is the volume of your cDNA synthesis reaction). Pay attention to the relationship of concentration, volume, and mass.
To better confirm the quality of total RNA extracted, I would suggest you to perform agarose gel electrophoresis and find the component of total RNA which consists of mRNA, tRNA, and rRNA. Find the expected size and location on gel image of the respective components online. If they are all in their expected location on gel image, you can safely assume that your total RNA is of high integrity. The spectrophotometry you performed only tells you about purity.
Usually this depends on the type of kit used and the company that produced it. Some companies require a higher amount of RNA to synthesize cDNA.
Carefully review the kit protocol you will be using to synthesize cDNA and follow the steps.
Before that, it is preferable to confirm the integrity of the total RNA by performing an agarose gel electrophoresis of the samples and RNA and watching the pands that confirm its integrity . If you do not find these pands , the RNA must be re-extracted again.
Dear Ali Ahmed Azzawri Win Sen Heng Ali Khalafizadeh Thank You very much for your valuable comments and time.
I think you want to convert 1ug of RNA to 1ug of cDNA in 20ul total reaction volume. If you want to convert more, you have to add more reagents. So, if 1ug RNA is present in 5ul, take 5ul RNA and other reagents and adjust the water to 20ul. If it is present in 10ul, take 10ul RNA and other reagents and take less volume of water to make the final reaction volume. So, it is the interplay between RNA volume (for 1ug) and water volume. You do not need to worry about what the volume is, concentrate on what the final amount is taken for cDNA conversion.
Dear Mr. Ahmad
by considering other people's points, please consider there is a possibility that you can use fewer amounts of total RNA based on the cDNA synthesis kit quality. for example, newly we found for super script III (invitrogen) in place of 1ug we can use just 250ng.
with best wishes
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