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RNAworld 11-02-2010 12:11 AM

Reverse Transcription (RT)
I am trying to get reverse transcription and dideoxy sequencing on my RNA. but since my RNA has strong secondary structure, the reverse transcriptase cannot get through it. I am using AMV at 52oC.

Also, I see a lot of background other than the full length product and unextended primers in my control lane, . anyone can help?

danfive 11-02-2010 04:20 PM

Re: Reverse Transcription (RT)

but since my RNA has strong secondary structure, the reverse transcriptase cannot get through it
That is a big assumption, and the least likely cause. The secondary structure can be there but really never seen it stop an RT-PCR.

reducing amount of RNA (likely cause of background)
Make sure RNA is best quality you can get (RNAse free, desalted, fresh)
1 primer only for transcription (low concentration so it gets used up)
reduce primer-dimer problem in PCR (reduce concentration of both primers, optimize #cycles, PCR-annealing temp, extension time etc).
After doing some or all of the above and if still in need of optimization:
you can pre-melt RNA (melting the 2nd structure really 95C for 30-50seconds) with primer (mixed/vortexed in RT rxn buffer only); very quick chill on ice, followed by adding rest of RT rxn mix and then beginning your RT rxn.
--A specific primer annealing should be facilitated and should improve your final results.

Good luck.

RNAworld 11-02-2010 10:20 PM

Re: Reverse Transcription (RT)
I will try that tomorrow! Thanks! will there be a big difference if i pre-melt RNA in water and in RT rxn buffer?

danfive 11-03-2010 01:31 AM

Re: Reverse Transcription (RT)

Originally Posted by RNAworld (Post 423551)
I will try that tomorrow! Thanks! will there be a big difference if i pre-melt RNA in water and in RT rxn buffer?

Use RT or PCR buffer, you need the buffer for quality control/protection from hydrolysis.

IHaveThePoint 10-14-2014 10:23 AM

Re: Reverse Transcription (RT)
See if this useful to you
RNA Extraction (“Invitrogen” TRIZOL Reagent #15596-018)
1. Add 3ml TRIZOL reagent in 40x16um frozen sections.
2. Mix and incubate at room temperature for 5 minutes.
3. Add 600ul chloroform to the sample. Shake vigorously by hand for 15 seconds and incubate at room temperature at room temperature for 3 minutes.
4. Spin at 12000rpm at 4˚C for 10 minutes.
5. Transfer the aqueous phase to the new tube.
6. Precipitate the RNA by mixing with 1.5ml isopropyl alcohol. Incubate at room temperature for 10 minutes.
7. Spin at 12000rpm at 4˚C for 10 minutes.
8. Wash the RNA pellet with 3ml 75% ethanol. Mix.
9. Spin at 7500rpm at 4˚C for 5 minutes
10. Air dry the pellet.
11. Dissolve RNA in DEPC H2O.
No mAb (-ve) cell line, check AutoF.
If –ve stained, continue keep it in NGS; If +ve stained, take out and end it by replace solution.

DNase Treatment of RNA
1. Prepare the reaction mixture: 10X DNase buffer 6ul, DNase I (10U/ul (2ul, RNase Out (40U/ul) 1ul, 10ug RNA + DEPC H20 51ul
2. Incubate at 37˚C for 30 minutes
3. Stop reaction by adding 6ul 0.1M EDTA (pH8.0)
4. Add DEPC H20 to 400ul, then add 400ul H20 phenol. Chloroform:IAA (50:49:1)
5. Mix and Spin at 12000 rpm at 4˚C for 10 minutes
6. Transfer the stop phase to 1.5ml eppendorf tube. Repeat steps 4 to 6 once.
7. Precipate RNA by adding 40ul 3M NaOAc and 800ul absolute ethanol.
8. Keep at -20˚C for at least 2 hours.
9. Spin at 12000rpm at 4˚C for 5 minutes
10. Remove supernatant and wash the pellet with 500ul 70% ethanol.
11. Spin at 12000rpm at 4˚C for 5 minutes.
12. Remove supernatant and dry the pellet.
13. Resuspend the RNA pellet in 15ul DEPC H2O.
14. Store at -80˚C.

10X DNase buffer 1M Tris-HCL (pH7.5) 400ul, 5M NaCl 20ul, 2M MgCl2 30ul, DEPC H2O) 550ul.

Reverse Transcription (‘Invitrogen’ SuperScript First-Strand Synthesis System for RT-PCR #11904-018)
1. Prepare DNase treated RNA/primer mixtures in sterile 0.5ml tubes as follows: 5ug total RNA + DEPC H2O 8ul, 10mM dNTP mix 1ul, Oligo dT (0.5ug/ul) 1ul.
2. Incubate at 65˚C for 5 minutes, then place on ice for at least 1 minute
3. Prepare the master mix: 10X Room Temperature buffer 2ul, 25mM MgCl2 4ul, 0.1M DTT 2ul, RNase Out (40U/ul) 1ul
4. Add 9ul of reaction mixture to each RNA/primer mixture, mix gently.
5. Incubate at 42˚C for 2 minutes.
6. Add 1ul (50 units) of SuperScript II Room Temperature to each tube, mix, and incubate at 42˚C for 50 minutes.
7. Terminate the reaction at 70˚C for 15 minutes. Chill on ice.
8. Flick spin. Add 1ul of RNase H (use your own set of pipetman) to each tube and incubate at 37˚C for 20 minutes.
9. Add 380ul sterile MQ-H2O to each tube.
10. Store cDNA at -20˚C.

Immunohistochemistry on paraffin section by Envision Kit
1. Cut 4-6um thick paraffin section on Tespa coated slide.
Dewax the paraffin sections in xylene (3 minutes, 3 times) and rehydrate through ethanol series (99% ETOH 3 minutes, 3 times); 95% ETOH 3 minutes and 80% ETOH 3 minutes
3. Rinse with distilled H2O 3 times
4. Antigen retrieval by pressure cooker method at 200˚C. Heat sections in 2L 10mM citrate buffer (pH6.0) for 5 minutes when the pressure reach at maximum.
5. Cool down the sections to Room Temperature.
6. Rinse with distilled H20 3 times
7. Remove endogenous peroxidise activities by treating the sections in 3% H2O2 70ul (63ul + 7ul) in H2O for 10 minutes at Room Trmperature. (Add 15ml 30% H2O in 135ml H2O)
8. Rinse with distilled H2O 2 times, then 1X TBS.
9. Add 1200 diluted primary mouse PTEN antibody (diluted in 1% BSA) to the section and incubate in a moist chamber at 37˚C for overnight.
10. Rinse with TBS 3 minutes for 3 times
11. Add a drop of anti-mouse antibody labelled with polymer+HRP and incubate at room temperature for 30min.
12. Rinse the section with TBS 3 minutes for 3 times
13. Develop the signal in freshly prepared DAB at room temperature. (1 drop DAB in 1ml substrate buffer)
14. Wash in tap-water.
15. Counterstain nuclei with Mayer’s Haemtoxylin for 1 minute.
16. Blue in Scott’s Tap H2O
17. Wash in tap-water, dehydrate, clear and mount.

10X citrate buffer (pH6.0), 1L: Citric acid 21g, NaOH 10.4g
20X Tris buffer saline (TBS) (pH7.6), 1L: Trizma 60.57g, NaCl 87.66g
BSA mix with TBS for blocking
BSA mix with TBS mix with antibody for dilution
Mili = m (10-3), Micro = u (10-6)

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