Any help? which reading to trust ? if trusted the Spectro results i will load around 1ul or less from my Rna sample for reverse transcription but if i used qubit results i will load 20ul of Rna WHICH ONE TO TRUST ?
Dear Basma Ali,
It is not clear what is the general purpose of your experiment.
But, I would propose you to chose and further work just with one of the equipment, and elaborate whole working path with the reference to it. If you must find real RNA content in all the samples, then better prepare dilution series of know samples (from yours or from another scientist who already use that RNA). check both equipments for the RNA content (and quality).
If you are not restricted to the RNA content, or reagent consumption, you should proceed with the working solutions measured by devices to revers transcription and check the results. But do it correctly, with several repetitions etc.
though I like Qubit, it has reagents to consume, while spectrophotometer don't, so is cheaper.
Good luck!
- The qubit for definite
- the spectrophotometer measures all aromatic rings at 260nM:
- In a pure solution of nucleic acid this will correspond to excitation of electrons in aromatic rings linked to nucleotides
- However, in impure solutions other aromatic based structures such as phenol, Trizol or free nucleotides can greatly increase the reading over and above RNA or DNA
- In contrast, fluorimetry associated with the qubit is based on flourescent dyes like sybr dyes that will only emit when bound to RNA; Readings therefore tend to be low the general UV spec as these other aromatic sources of emission are not detected
- Therefore trust your lowerr ubit reading - which is associated exclusively with RNA - and not the higher spec reading which is evidently picking up contaminants as well, e.g. Trizol or phenol for manual RNA extraction or Aromatic salts associated with kit based lysis and denaturing buffers, cf. GITC
thank you so much Dr Laurence Stuart Dawkins-Hall , one more question if in my lab qubit kit is not available in high quantities to be able to measure all samples , can i measure like 2 -3 samples by qubit and extrapolate the measure for my other 16 samples .
i mean i need to reverse transcribe (1 microgram from each sample ) qubit will help me to find how many micro liters to take. can i relay on endogenous control to account for any error ?
- You really need to be able to quantify each sample exactly so know you cannot extrapolate
- This time you will need to take all your samples on ice and measure with a qubit:
- Then reverse transcribe exactly 1ug and take exactly 1ul of cDNA for each sybr green qPCR reaction
- Going forward you need to purify your samples more effectively so that measurement by Spec @ 260nM is not comprimised by imurities (that add to reading compared to qubit):
- If you are extracting RNA using trizol then this will add strongly to your spec reading @ 260nM unless completely removed:
- The best way to do this is to perform 2 x chloroform extractions after your Trizol/Phenol extraction; and then precipitate the upper aqueous phase from your chloroform extraction by
- Crucially if the above removes most of the contaminating phenol/Trizol your 260/230 ratio will be < 2.2 and also your 260/230 ratio will be > 1.0 ( < 0.5 is large phenol contamination
- If extractions are performed properly as described, you should be able to measure your RNA by spec; RT 1ug and then take 1ul of each cDNA replicate for qPCR; I do this routinely
- Note: YOU CAN NEVER measure cDNA on a spec as the reading will almost exclusively consist of dNTPS that have not been incorporated in the cDNA (from RT reaction mix). In this case you either purify away your dNTPs (using Qiagen spin a column) in which case you can then use a spec or you can use a Qubit which will only measure your cDNA and not excess dNTP monomers.
- You then put exactly 1ug of cDNA measured by Qubit into your qPCR reaction
- This is the aternative to making intial measurements of RNA with spec; performing RT with 1ug of measured RNA and then using 1ul for all samples in qPCR but it offers no real advantages to spec measurement by RNA as differences in cDNA reactino efficiency are normalised by your housekeeping genes
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