I have tried so many times to extract tRNA from Rose leaves and buds by pBiozol method. My intention is to have tRNA for experiments about miRNAs (as pBiozol can give tRNA with high concentration of miRNAs). Here is the customized protocol that i follow (the steps indicated by * are added by my lab's senior members):
1) crush tissues in liquid N2
2) add pBiozol: 0.5ml for 0.1g tissue, 1ml for 0.2g, 5ml for 1g, and so on.
3) incubate on ice (horizontal placed tubes) for 5min
4) spin @12k g for 2min at 4oC
5) transfer supernatant to a new tube; add 0.1ml NaCl (5M) and 0.3ml chloroform (if 0.1g tissue used in step 2)
6) spin @12k g for 10min at 4oC
7*) transfer the supernatant (uppermost phase) to a new tube; add the same amount (as of supernatant) of phenol:chloroform at 25:24
8*) spin @12k g for 10min at 4oC
9*) transfer the supernatant (uppermost phase) to a new tube; add the same amount (as of supernatant) of chloroform
10*) spin @12k g for 10min at 4oC
11*) transfer the supernatant (uppermost phase) to a new (final) tube; add 1ml of isopropanol alcohol and incubate at -20oC for 2 hrs
12*) spin @12k g for 30min at 4oC
13*) discard supernatant, add 80% ethanol
14) spin @12k g for 10min at 4oC
15*) repeat step 13 and 14
16) remove all the ethanol, air dry pellet for a few minutes, add DEPC water 20-30ul, store at -80oC
17) check quality and quantity by nanodrop and gel electrophoresis
(all the chemicals used are pre-chilled; tubes are worked on ice; extra care for avoiding contamination).
I use young leaves or flower buds. I just assume the weight of tissue as 0.1 or 0.2g (may be i am wrong, but how can i weight the tissue before adding the pBiozol, it seems impossible to me)
now the question is, where is the problem? i mostly get no bands, smeared band, or dim 28s band..
Please help me, i have tried more than 30 times
Hi Muhammad,
1) To answer your question: The easiest way to measure your tissue's weight is the following:
1. Put a fresh, empty, closed tube with sample ID label onto the balance, than tare it.
2. Remove the tube from the balance. Put the tube in cold evironment
(preferably onto dry ice).
3. Do the snap-freeze process of the plant tissue with LN2 then transfer the frozen sample into the cooled tube.
4. Put the tube with sample in it onto the tared balance. Than it measure the sample's weight. Put back your tubes onto dry ice until you can begin lysis buffer addition.
5. Repeat this process with all of the samples.
2)
I think the problem is lenght of the time between the sample snap-freezing with LN2 then lysis buffer addition. Try to reduce to minimal time between these steps. RNases are really stable and high activity enzymes, so they need only litlle time to disrupt RNA.
Or might can be problems with the efficiency of the lysis. The plant cell's wall can be hard to disrupt. You can try mechanical methods (steel beads or steel blade disruptors to homogenize your frozen plant tissues with your lysis buffer (pBiazol).
I have got many years of experience in RNA isolation. Please let me to note some suggestions to you.
The Trizol or any similar acid-guanidin-isothyocianate-phenol based lysis buffer effectively inhibit RNases in your samples. The main point is to add the Trizol immediatelly to your sample after thawing followed homogenization by pipetting the cell pellets up and down for severeal times. Then use the classical phenol-chloroform-isopropanol purifing method. I've read that pBiozol is more than GITC/acidic phenol based lysis buffer (beside the earlier compounds it consists ammonium-tiocyanate and 2-mercaptoethanol also - later can irreversibly denature RNases by reducing disulfide bonds and destroying the native conformation required for RNase functionality.
Alternatively you can use the Trizol lysate directly in Direct-zol RNA spin column kit https://www.zymoresearch.com/media/amasty/amfile/attach/_R2060_R2061_R2062_R2063_Direct-zol_RNA_MicroPrep_ver._1.0.0.pdf). I have very good experiences with this kit in human chordoma tissue RNA isolation in the terms of RNA quality and quantity. I have used the Trizole lysate after chloroform phase-separation step in the above kit. To decrease the protein contamination in the further purification steps
GITC/acidic phenol lysis combined with silica-based column methods
(Pros: after phase separation step no need to use chemical hood, can be high throughput Cons: more expensive than GITC-based methods
GITC/acidic phenol methods
(Pros: the cheapest, intact total RNA (including rRNAs, miRNAs) can isolated with high yield, Cons: toxic organic solvents (phenol, chlorophorm) (need a chemical hood), very problematic to use in high throughput).
Bests,
Tamas
Thats so nice of you Tamas Kerekes. Thank you so much for the detailed answer. I will follow your suggestions. Highly appreciated
Still no success :`(... I found these results in Arabidopsis and Tobacco, so my professor said Rose RNA might be very difficult to isolate :`( ... i don't know what to do ..... By the way I take a lot of care in each and ever step. In the weighing part, I pre-weigh the tubes with steel beads and write that weight on the tube. Then i collect samples in these tubes and right away throw them into LN2, then weigh them and calculate the sample weight, and then add the lysis buffer accordingly. But before adding lysis, i mechanically grind the samples within the tubes, the steel bead do its part and thoroughly change it to fine powder. My professor said this is wrong, you should grind the sample by motor and pestle, but whenever i do that, the sample is not grinded sufficiently.
I will try the Trozol lysate as you mentioned. Hope it goes well.
I figured out what the problem was.. The damn protocol is wrong. The stupid manufacturer did many mistakes in it.. Most importantly, the amount of tissue needed is far more than the actual one. That is so ridiculous
Hi Muhammad,
Sorry to hear that. To tell the truth I've been ran into that problem many times with factory protocols (e.g.: Direct-zol RNA spin column kit's reagenst amount has errata in the protocol. So I had to recalulate the amouns, otherwise it hasn't fit in the tube). At least people are working in factories also. They can make mistakes. :S But I know that it's very annoying. I hope that finally you can isolate good quality and intact RNA from your samples.
Bests,
Tamas
Sorry to start this thread again, but I have a question.. Since Rosa tissues have lot of Phenolic compounds that makes its RNA difficult to isolate, does Phenol:chloroform purifying method makes any sense in the protocol, or just chloroform purification is enough?
i extracted total RNA and somehow i got good bands. Then I used DNAse1 to remove gDNA, it failed completely, erasing the RNA bands while gDNA was still there. Then I used reverse transcription to make cDNA. I checked it with UBI, there was no cDNA at all. Don't know whats the issue.
One thing that irritates me is the color of RNA pellet, that is still prominent in the diluted RNA, and cDNA as well. May be the problem is that RNA has some impurities, or it isn't washed completely. What do you experts say?
Hi Muhammad,
DNAse I can cleave only ssDNA or dsDNA, nut not RNA. Therefore the majority of your bands seem to be DNA despite of RNA.
I suggest to check your samples with separate nucleic acid stains (DNA and RNA) to reveal the correct amount of them. E.g Invitrogen Qubit System or any alternative.
I think your RNA isolation method not the best effective. I have told you in my earlier answers. Rosa tissue cells can be difficult to lyse because of the cell walls. Try mechanical lyse methods (stainless steel blade distruptor).
I think the GITC/acidic phenol methods hasen't affected by the phenolic compounds of the Rosa tissue.
Alternatively you can use the Trizol lysate directly in Direct-zol RNA spin column kit https://www.zymoresearch.com/media/amasty/amfile/attach/_R2060_R2061_R2062_R2063_Direct-zol_RNA_MicroPrep_ver._1.0.0.pdf). I have very good experiences with this kit in human chordoma tissue RNA isolation in the terms of RNA quality and quantity. I have used the Trizole lysate after chloroform phase-separation step in the above kit. To decrease the protein contamination in the further purification steps. But It can expensive compare to pBiazol method.
Bests,
Tamas
yes Sir you are right, but i don't have any other options.. I have tried a lot for the Direct-zol_RNA_MicroPrep kit, but the lab doesn't agree to provide me finances, otherwise i would have been successful long ago...I have to rely on the old conventional methods.. Most of the reagents i use are outdated, but still no hope for replacements..
Anyhow, thank you
Hi,
Unfortunatelly I know the "lack of money but save the world" situation. Tomorrow I'm going to send you the my TRI reagent (similar to pBiazol) protocol. in English. It can worth a try. You need the almost the same reagents. Please try. It based on the classical TRI Reagent (MRC) (e.g. Trizol and other GITC/phenol-based lysis buffers) protocols.
Bests,
Tamas
sir, i have tried TRI reagent with different plants, and it is not compatible with Rosa, no extraction at all
Hi Mihammad,
I guess you have tried MRC TRI Reagent original protocol (chloroform phase separation followed by isopropanol precipitation) for the RNA isolation atempts. Check the attachment please.
Tamas
what about CTAB+PVP for remove polysaccharides and polyphenol ？
maybe this paper will help you
Yes sir, I have tried this method and got good results on the gel, but my professor said it isn't suitable for downstream miRNA applications
at last，which method is suitalbe for your downstream miRNA applications ？
In order to continue my work flow, I had to skip this experiment (extraction by pBiozol reagent) as it wasted a lot of time, I will have a fresh start probably next month
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