For isolation of cytoplasmic RNA from animal cells or eukaryotic cells, we recommend Total RNA Extraction Kit (Blood/Cultured Cells; RB).You will have to prepare an extra buffer (not included in RB Kit) to lyse plasma membrane before going on the regular RB protocol.
Plasma Lysis Buffer Components: (Pre-cool to 4°C )
50 mM Tris-Cl, pH 8.0
140 mM NaCl
1.5 mM MgCl 2
0.5% (v/v) Nonidet P-40 ( 1.06 g /ml)
Just before use, add:
1000 U/ml RNase inhibitor
1 mM DTT
Add Buffer to lyse plasma membrane:
For pelleted cells, loosen the cell pellet thoroughly by flicking the tube. Carefully resuspend cells in 175 µl cold ( 4°C ) Plasma Lysis Buffer, and incubate on ice for 5 min.

Users could use both Total RNA Extraction Kit (Blood/Cultured Cells) and Total RNA Extraction Kit (Tissue ) for RNA extraction from yeast .

Please follow this modified protocol.
A. Harvest yeast cells (up to 5 x 10 7 ) by centrifugation for 10 min at 5,000g .

Discard the supernatant and resuspend the pellet in 600 µl of sorbitol buffer.
B. Add 200 U Lyticase or Zymolase. Incubate at 30°C for 30 min. Centrifuge

the mixture for 10 min at 2000 g to harvest spheroblasts.
C. Remove supernatant and go to step 5 of Bacterial Cell Protocol (RB)

The extra filter columns are effective in clearing cell debris and ensuring full cell lysis.

No, some genomic DNA (and plasmid DNA, if present) can be co-purified with RNA. DNA can be removed by adding RNase-free DNase I to the RNA sample. DNase I can then be removed by phenol/chloroform extraction.

Three critical steps, if they are not done well, RNA degradation may occur.

They are (1) handling and storing of samples, (2) disruption of samples, (3) storage of eluted RNA

(1) Most animal tissues can be processed fresh (unfrozen). It is important to keep fresh tissue cold and to process it quickly (within 30 minutes) after dissecting. If samples cannot be processed immediately, it should be flash frozen in liquid nitrogen and stored at -80°C . Samples should be handled with RNase-free tools.

(2) When sample is disrupted, disruption needs to be fast and thorough. Slow disruption, e.g. placing cells or tissue in RB Buffer without any additional physical shearing, may result in RNA degradation by endogenous RNase released internally, yet still inaccessible to the protein denaturant in RX Buffer.

(3) After elution of RNA with RNase-free ddH 2 O provided in the system, store RNA at -80°C .

(4) Degradation of RNA may also occur during loading into a gel, use gel and fresh running buffer prepared using DEPC-treated ddH 2 O as well as properly cleaned gel tray and tank for electrophoresis. Adding EtBr directly into the gel can also avoid possible degradation of RNA that may occur during gel staining.

(1) Poor yield of total RNA is mostly due to incomplete sample lysis, thus leading to incomplete release of RNA. Since good yield and good quality of total RNA are only assured when sample is properly handled and lysed completely, do not use more than the amount of sample suggested in the protocol.

(2) Thorough cellular disruption is critical for high RNA quality and yield. RNA that is trapped in intact cells is often removed with cellular debris and it is unavailable for subsequent isolation. Therefore it is crucial to choose the disruption method best suited to a specific tissue or organism to maximize yield. Mechnical cell disruption techniques include grinding, homogenization with douce or rotor-stator homogenizers (polytron), vortexing, sonciation, and use of bead and freezer. Complete disruption of some tissues may require using a combination of these techniques. Rotor-stator homogenizers, alone or in conjunction with other disruption techniques, generally result in higher RNA yields than other types of homogenizer.

(3) Another most frequent cause of low RNA yield is overloading the column, which can cause the column to be clogged or can prevent the RNA from binding to the membrane efficiently. Methods that reduce viscosity, such as reducing sample amount, disrupting the sample more extensively, and centrifuging to remove insoluble remains, will increase RNA yield. If yields are still lower than expected, consider diluting the clarified lysate and splitting loading into two columns, which will further reduce the concentration of contaminants and improve RNA binding and recovery.

(4) When RNA is to be eluted, make sure that RNase-free ddH 2 O is added onto the membrane and penetrate into it. If ddH 2 O still retains on the membrane, pulse centrifuge the column for a few seconds to drag it into the membrane.