Dear Helmi

• I have never run bleached gels for denaturation but I have run standard agarose gels as well as denaturing agarose gels in the past and in truth all of your RNA looks degraded;
• that is I can visualise what I think is the bottom 18s Ribosomal subunit but not the top 28s rRNA sub unit
• This will be about handling extraction and storage of your RNA.
• If you provide details of exactly how you Trizol extract including harvesting of cells I might be able to help
• I have harvested human cells including cancer cell lines in the past by Trizol extraction

Thanks Laurence and Mohamed for your response.

Yes, I check the RNA by the nanodrop, and the concentrations were over 500ng/ul and the 260/280 over 1.8.

regarding to the steps I followed for the Trizol extracting method.

• Started by adding cold PBS to the 48 hr old cells then removed the PBS
• Added the 3 ml Trizol, incubated 3-5 mins to homogenize and scrape the cells
• Transferred the cells to the microtubes (1ml each), added 200ul chloroform to each, vortex for 15 sec. and centrifuged 15 mins at 12,000xg at 4 degree.
• Collected the aqueous phase in new microtubes, added 1 ml isopropanol, incubated the samples at 20 degree for 10 mins and centrifuged at 12,000 xg for 10 mins. at 4 degree.
• Removed the isopropanol and RNA washed by adding 1ml 75% ethanol once. at 7,500 xg for 5 mins at 4 degree.
• After that I removed the ethanol from the tubes and left the RNA tube on the bench for drying (the drying time was quite long 20-30 mins) because of the remaining amount of ethanol.
• And the RNA dissolved using RNase DNase free water (but not DEPC-treated water).
• The concentration and 260/280 were checked on nanodrop.
• Then stored at -80 for two weeks until I took them to check on bleached gel.

Check the A260/280 ratio with spectrophotometer (e.g. NanoDrop), the number indicates contamination by protein or reagents. ~1.8 is accepted as pure for DNA, 2.0 is for RNA. There is also A260/230 to look contaminants during the extraction procedure. 

Dear Helmi

1. Leaving the pellets for 30 minutes @ room temp for RNA is not advisable. Although RNA is relatively stable in pelleted form, I would

• Add the isopropanol
• Split between 2 x 2ml screw cap tubes
• Spin at 13,000 rpm @ 4C in a microfuge for 20 min
• Remove most of the ethanol supernatant with a 1 ml tip
• Add back 0.5-1ml (per tube) of 70% ethanol
• If pellet visible, vortex to dislodge and more effectively desalt
• Spin pellet again for 5 min @ 13 K @ 4C to secure on bottom of tube
• Remove supernatant with a 1 ml tip to just above the pellet
• Remove supernatant against the pellet itself with a p20/200 tip
• NOW SPIN AGAIN for 1 min to bring the residual ethanol on sides of tube down and remove with a p10 tip: This will render the pellet essentially dry
• Thus you can dry for dissolving by placing ON ICE for ~ 15 minutes and then at bench for 5 minutes to completely dry; at which point the pure RNA will turn transparent
• Dissolve as you describe but dissolve on ice with perdiodic pipetting up and down before placing @ -80C

2. If you cannot spin @ 4C in a refrigerated microfuge and thus use a sorval:

• Add your 70% ethanol,vortex spin for 5 minutes, pellet as describe, remove supernatant as described then spin again for 1 min to remove residual ethanol with a P10 tip

3. Regarding 260/280 and 260/230 ratio your 260/280 ratio is good but pay attention to your 260/230 ratio as well which should be > 1.0

4. < 1.0 indicates Trizol and/or Guanidinium salt contaminants and these can inhibit down stream reactions: During precipitation, the 70% wash is designed to denude the pellet of these contaminants and render the 260/230 ratio > 1.0 and vortexing if you can see the pellet to dislodge will render this was more effective and thus render your 260/230 ratio > 1.0 more of the time

5. Also, do not confuse the quality of your RNA indicated by the above spec indexes with integrity: You can have decent 260/230 ratios but still incur degradation: Try to avoid putting RNA at room temp for prolonged periods to minimise degradation (as well as use molecular grade reagents as you describe

6. Also, make sure your Trizol is < 6 months old especially if it has been stored in the fridge: Old Trizol can oxidise and resulting quinones can degrade both DNA and RNA

Hi Laurence, Thanks for your notes. I repeated the RNA extraction with a lot of care and I took into consideration your recommendations such as the 13000 for 20 and less drying time and the process went good with me. Please see the attached photo and let me know your comments.

thanks again.

Dear Helmi

• I am pleased to report that your RNA looks intact: I think I am right in saying that your 2 distinct bands are 2 x integral rRNA subunits; What is more the upper band is stronger than the lower rRNA band and this is a good sign of lack of degradation:
• In a perfect scenario your 28s rRNA to 18srRNA subunits derived from mammalian cells should be present at a ratio of 2:1
• As long as you can see those 2 bands as distinct entities without smearing between or below those subunits however that is a sign that your RNA is sufficiently un degraded that it will work in functional assays
• Completely un degraded RNA manifests a stronger upper band (28s) which yours appears to show !!
•  Significantly degraded RNA that often doesn't work in functional assays invariably shows just the lower 18s rRNA subunit with much degraded (smeared) material below

Hello Laurence,

I really appreciate you disscussion and your comments on my results. I used the same RNA for qPCR and I experienced not good melting point! Can you please check my new question in the link below. It would be very helpful if you could comment on that and recommeneded some trouble shooting. Thanks 

https://www.researchgate.net/posta/Melting_curve_What_the_factors_that_may_affect_on_melting_curve