A couple of suggestions,

For your sample in Trizol, use a normal protocol to isolate RNA but during the precipitation (ppt) step add Glycogen (RNase free). Generally 5ug is enough. Glycogen is inert and doesn't interfere in various down stream applications (such as PCR, sequencing, RT etc) up to a concentration of 8mg/ml.

I am not aware of any method (or an initial step) to condition your silica column to increase the yield in column based methods of RNA isolation. It may be possible.

For your samples in B-ME, I would suggest you to add glycogen to your samples before adding it to the column. Incubate at RT for 5 min, proceed to your RNA isolation method. After washing and other steps, elute your RNA in 2x100ul in RNase free water. Then again ppt your RNA using 1/10 vol of 3M Sodium Acetate+2.5x vol of 100%Ethanol. If you want you can again add glycogen during this ppt step. Another thing which may help in getting higher nucleic acid yield is to incubate your samples at -80°C overnight during the ppt procedure.

But remember, both of these methods will also co-purify some quantities of genomic DNA. So whatever yield you get will also contain some DNA, if you don't perform DNA digestion.

Good luck.

Agree with Dr. Sandeep Gupta. If you have insufficient number of RNA or original cells you shoul use carrier (glycogen) during isolation. You can achieve good results with RNeasy Micro Kit, RNeasy Plus Micro Kit (Qiagen).

If you confident in the purity of the cells samlpe you may modify ambion protocol:

1. add in sample with lysis  1 volume 85% Et instead of 70%Et before transfer to the column,

2. eliminate washing step with washing buffer 1. So use only 2 washings with Washing Buffer 2 (WB2 needs 85% Et instead 70%!)

3. DNase treatment needed!

These modifications will help to reduce RNA loss during purification.

Thank you very much for your input Dr. Gupta, Dmitry.

I tried all the methods you both mentioned using the kit I already have but I was only able to retrieve 80ng of total RNA from the small amount of cells I had. I am borrowing a different column for tiny amounts of cells (the qiagen micro kit) from a neighboring lab, I will update this thread when I get a successful result!

Alice, 80 ng of total RNA is quite good result for sample with only 300-700 cells!

There are about 50 pg of total RNA in single mammalian cell.

(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3346307/pdf/jove-53-3144.pdf)

Anyway, try Qiagen micro kits. I think they are the best ones.

Agree with both comments above.  Another way is to combine chemical extraction method (phenol/guanidine salts, or Trizol method) with solid phase extraction method (immobilization glass filter embedded spin column, or silica columns).  Silica spin column is used to replace the ethanol precipitation and you don’t need to use glycogen or other RNA carrier.  Below is the procedure.  In your case, since you want to isolate RNA from very small sample, then it is better to use Tini RNA spin column (EZC107).  This tiny column was originally designed for DNA tiniprep from single colony and DNA gel extraction (agrose gel).  So, you can do tiniprep from single colony directly from LB agar plate without growing 2ml E.coli culture for DNA miniprep. This is the smallest column on the market and the elution volume can be as low as 3ul, so it is very useful for DNA/RNA isolation from very small samples and it can be used for DNA/RNA concentration as well. Our Tini spin column contains a silica membrane and it is compatible with all the solutions from any commercially available kits using silica based columns (e.g. Qiagen RNaeasy kits...).  Since it is a very small column, you only need to use 1/3 of solutions that for standard mini spin column.  Here are the links for our bulk tini and mini spin columns.  You can also order through ThermoFisher:

Tip: Buy bulk solutions from Qiagen and use our bulk columns to assemble your own DNA&RNA kits

http://www.enzymax.net/columns/tini_spin_DNA_column.htm (DNA Tini column: $33, Fisher #NC1062436)

http://www.enzymax.net/columns/tini_spin_RNA_column.htm (RNA Tini column: $49, Fisher by request)

http://www.enzymax.net/columns/mini_column_DNA.htm (DNA mini column: $38, Fisher #NC1013737)

http://www.enzymax.net/columns/mini_column_RNA.htm (RNA mini column: $59, Fisher #NC1062437)

Procedure:

1. TRIzol/ chloroform extraction as usual.

2. Carefully transfer the supernatant to a clean tube and measure the volume.

3. Add 1-1.25 volume ethanol (95-100%) to the supernatant. Mix by inverting the tube several times.

Note 1: This step is for recovering total RNA including small RNA>17nt.

Note 2: If recovering the RNA>200nt, add equal volume of 70% ethanol (instead of 1-1.25 volume) to the supernatant.

4. Load the mixture to the column with collection tube and spin for 1 min at 40C at 12,000xg. Discard the flow-through.

5. Add 350μl of 80% ethanol (v/v) and centrifuge for 1 min. Discard the flow-through. Centrifuge the column for an additional 2 min to remove the excess ethanol. Note: this step is to remove the residual ethanol.

6. Elute the RNA by adding 3-20μl of DNase/RNase free water (pre-warmed at 650C) to the center of the Tini spin column (or 50ul for mini spin column) and incubate at room temperature for 5 min. Centrifuge at max speed for 1 min. The eluted RNA can be used immediately or store at -800C.