I am trying to construct a cell line library for yeast teo hybrid mating. the mRNa concentration was low so I concentrated using a vacuum concentrator and the final conc. of mRNA was 370 ng/µl. I am using this as the source for constructing library using Mate and plate library system (CLontech). However I could not get anything on LD PCR. Previously 6 months ago I made one library using the same system. I got the dscDNA smear upto 1.5 kb so I did not proceed further as its being suggested that smear of ds cDNA should be around 3 kb. But this time I am not getting dscDNA. I checked the sscDNA by doing PCR of beta actin.
Hello Sonalika,
A few questions:
1. Do you have a limited amount of the cell line? If not, it would be worth isolating your RNA and checking the integrity of the resulting RNA by visualizing it on the standard denaturing gel. Have you already accomplished this? If so, what did you observe?
2. Assuming that your RNA is intact prior to vacuum concentration, you might want to check the impact of this concentration step on your RNA integrity.
3. After vacuum concentration, do you wash your RNA in 70 % ethanol to remove salts that would have concentrated with the RNA and that would reduce the efficiency of steps that follow?
I will be able to provide better advice once I know the answers to these questions.
All the best
Hello Federa and thanks for ur suggestions
1. i have checked the integrity of the RNA on Formaldehyde denaturing gel. for the RNA, extracted from naive cell line, 28s and 16s bands are prominent. however in RNA infected from virus infected cell lines, there are multiple bands of equal intensity (thiugh 28s and 16s bands are prominent too).
2. i have not check the impact of concentration step on RNA integrity, neither I wash teh RNA after concentration in 70% alcohol. can you please sugeest the pre and post concentration steps in little detail.
Another thing happening with me is that the results in first strand cDNA synthesis and LD-PCR are not repeatable. I mean sometimes RT is failing and sometimes LD-PCR. once I have double strand cDNA , i check it in a 50µl reaction initially and the smear was upto 3 kb but on repeating in bulk (two 100 µl reactions), smear decrease to 1 kb.
Another thing is could u please tell me any protocol for mRNA concentration coz I’m getting around 50-80 ng/µl only and the maximum volume as mentioned by clontech is 2µl only.
OK Sonalika,
1. The prominent 28S and 16S bands indicated that your RNA is intact and therefore integrity is less likely the problem.
2. The fact that the first strand cDNA synthesis and LD-PCR are not repeatable suggests that there may be an inhibitor which co pellets with your RNA and varies depending on the concentration of the RNA.
3. What protocol are you using for the RNA isolation? I need to know this first before I can provide the best recommendations.
4. Without further information, I would suggest that once you have isolated the RNA and before using the concentrator, you can precipitate the RNA again by adding 1/3 vol 3M NaOAc pH 5.5, mix, add 2.5 to 3 vol ethanol (if your tube allows, otherwise you can keep the volume low by using 1 volume isopropanol) mix and incubate at -20 C for 30 min to ON (depends on the amount of RNA present).
If you have a high concentration of RNA, you will observe the solution become very cloudy as the RNA rains out of solution (you can centrifuge within 10 min). If the RNA concentration is low, then the solution will not become cloudy. In that case, you will want to leave it at -20 C for at least 30 min.
To pellet your RNA, the standard centrifugation at 10,000 -20,000 g for 10 min will work. Note the presence and location of your pellet in the tube. Discard the supernatant fluid without disturbing the pellet.
If you are not in microcentrifuge tubes, now is a good time to transfer to such tubes. To accomplish the transfer without significant loss, resuspend your RNA pellet in lysis buffer containing all the RNase inhibitors and transfer the solution to the eppendorf tubes. Reprecipitate with isopropanol as described above.
If you are already in an eppendorf tube, then once the supernatant fluid is removed,
add about 500 ul 70 % ethanol, vortex vigorously (RNA is not susceptible to mechanical damage as much as genomic DNA, therefore you can vortex), recentrifuge at 10K -20K g for 5 min. Note location of your RNA pellet in the tube. Carefully remove the supernatant fluid. Re-centrifuge your tubes for a couple of seconds, carefully pipette off any remaining liquid, let air dry or vacuum dry (about 2 min). Depending on the size of the pellet, you can choose the volume to resuspend the pellet. Start with no more than 20 ul (20 mM Hepes pH 7.2, 1 mM EDTA). This concentration of EDTA will not inhibit down stream reactions. If you need to dilute further you can use 20 mM Hepes pH 7.2 without EDTA. However, your stock should have buffer and EDTA.
Good luck and keep in touch
As per my experience with this kit you can directly use the total RNA for cDNA construction. But take care that for the cDNA construction, use freshly isolated RNA dissolved in freshly autoclaved MQ and not the DEPC treated as DEPC(even in small amounts if remains in MQ with hinder the reaction). Second thing is that carry out the RT reaction (where SMART oligo will be attached at 5' end of cDNA/template switching) in air incubator at 42 C rather than a water bath or PCR. I have worked with this kit several times and total RNA works superb.
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