Hello Mohiraa Shafreen

In plants, polysaccharides and polyphenols are two classes of biomolecules that vary significantly between species and become problematic when isolating RNA. Contaminating polysaccharides and polyphenols can cause interference during RNA isolation.

CTAB (hexadecyltrimethylammonium bromide) is a cationic detergent that facilitates the separation of polysaccharides during purification while additives, such as polyvinylpyrrolidone, aid in inactivating polyphenols. I would suggest you use CTAB extraction buffer to isolate RNA from plant leaf which is commonly used for nucleic acid isolation from polysaccharide-rich plants, and in particular RNA isolation.

A typical CTAB extraction buffer contains CTAB, polyvinylpyrrolidone (PVP), sodium chloride, and beta-mercaptoethanol, each of which plays an important role in nucleic acid extraction from polysaccharides. CTAB acts as a strong detergent to help to break plant cell walls and separate nucleic acids from polysaccharides. Salts help to dissolve polysaccharides and CTAB-RNA complexes, resulting in the efficient removal of polysaccharides and CTAB during chloroform extraction. PVP helps to prevent the oxidation of polyphenols in cell walls and extracellular matrices. Beta-mercapto ethanol is a strong reducing reagent that acts to irreversibly denature RNases.

Prepare CTAB Extraction Buffer as follows.

2% CTAB,

2% PVP40,

25mM EDTA,

100mM Tris at pH 8,

2M NaCl

Autoclave the extraction buffer and then add 1% (v/v) β-mercaptoethanol prior to extraction procedure. The final solution is warmed in a dry bath to 65 °C before use in RNA extraction.

You may follow the protocol given below.

1. The leaf sample which is pre-cooled in liquid nitrogen may be crushed into fine powder in a pulverizer.

2. The sample powder may be transferred to RNase-free microcentrifuge tube filled with 600 µl pre-warmed CTAB extraction buffer and vortexed for 5 min at room temperature.

3. Centrifuge at maximum speed (15000 x g) for 10 minutes and transfer supernatant to a new microcentrifuge tube.

4. Add an equal volume of chloroform: isoamyl alcohol (24:1), then vortex for 30 seconds and centrifuge at maximum speed (15000 x g) for 15 minutes at 4°C.

5. Pipette out aqueous phase, without disruption of the white interphase, and transfer to a new microcentrifuge tube.

6. Repeat the chloroform: isoamyl alcohol extraction as above.

7. Add an equal volume of ethanol (95-100%) to the aqueous phase, mix and load onto the spin column. Continue with the kit’s manufacturer protocol, and the resulting RNA may be stored at −80 °C.

Best.

I have used a similar protocol in the past with leaf samples that had a lot of secondary metabolites and it worked quite well. You can add a heat block incubation step too if needed, this can help dissolve the unwanted materials. After you add your leaf powder to the CTAB buffer, heat for 20 minutes at 65 degrees. If you heat the samples you'll either need locking tubes or to vent them periodically. Or, simply use a pin to poke a small hole in the tube lid. I would suggest putting the heat block in the fume hood just in case any tubes do pop open. Then proceed with centrifugation.

I use nucleospin blood RNA kit. Maybe we have the same problem : the impurities after lysis of cell can not be eliminated. These impurities will cover the DNA/RNA binding membrane in column and decrease the yeild. Here's my solution: before add the ethanol(70% or 100% ethanol, this is to adjust RNA binding conditions) to the tube contained RNA solution, centrifuge ~3min at ~14000rpm to settle the impurities to the bottom of the tube. Then pipette the supernatant(do not touch the sediment ) to a new tube and add ethanol. MIX and add to RNA binding column. If it‘s not OK, try use 2×or more solution and column to extract。