4.4　 RNA Extraction

Tomomichi Fujita and Mitsuyasu Hasebe

A. Precaution

(1) Use gloves, disposable tubes (usually not necessary to autoclave, use RNase-free tubes)

(2) Make a distinction between RNA utensils including regents and others (Do not use RNA utensils for other purpose. It is better to make special shelf for RNA regents)

(3) Do not open the caps of the bottles of RNase and Pronase in the laboratory.

(4) Do not weight RNase, DNase, and Pronase.　 Believe the label and dissolve the contents at a time with appropriate buffer.

(5) Do not discard high concentration of the solution or used tubes in the lab.

(6) Be careful for contamination. Use species-specific motor and pestle if necessary.

(7) Plant tissue is the biggest source of RNase, and mix tissue with buffer as quick as possible.

(8) You yourself are another source of RNase. Keep your mouth shut without having spit.

B. Pretreatment

(1) Sterilize all of utensils by autoclave (120˚C 20 min) or oven (200˚C 3 hours). Plastic tubes are not necessary to pretreat.

(2) Inactivate RNase with DEP (Diethylpyrocarbonate):

All of reagent except Tris

　　　　　　　　　　　 Add 2% (v/v) DEP in Draft

　　　　　　　　　　　 Keep at least 2 hours at room temp in Draft

　　　　　　　　　　　 Autoclave (121C 40min)

In case of Tris

Use RNase free reagents (usually no problem to use regular grade Tris, but do not share the bottle with other purposes)

　　　　　　　　　　　 Dissolve in DEP treated H2O with sterilized utensils

　　　　　　　　　　　 Adjust pH with pH paper or clean pH electrode

1）RNeasy (QIAGEN) --- for protonemata: for RACE, RT-PCR

2）ISOGEN, ISOGEN-LS (NIPPON GENE, Tokyo, JAPAN) --- for protonemata, sporophytes: for RACE, RT-PCR

3）GuSCN and CTAB method --- for protonemata: for northern, RT-PCR

4）DYNABEADS mRNA DIRECT kit (DYNAL) --- for both protonemata and gametophores: for any purposes

You can extract relatively pure total RNA from protonemal tissue, while it may be rather difficult to get pure RNA from gametophores. During RNA isolation steps from gametophore, polysaccharide-like moldy contaminant co-precipitates with extracted RNA after centrifugation, and this pellet never dissolve. Centrifugation is not involved in the method 4), and is useful to extract RNA from gametophores. Total RNA from young sporophytes with archegonium was successfully extracted by methods of 1) and 2).

We use different RNA extraction method dependent on purposes.

For RACE

Total RNA and mRNA extracted by 1) and 3) work for 3'RACE System kit (Invitrogen) and Marathon cDNA amplification system kit (CLONTECH). Total RNA by 3) may sometimes have troubles because of purity.

　

For Northern hybridization

It should be better to use about 1 µg poly(A)+RNA par lane for northern blotting, although 10 µg of total RNA par lane may work.

For RT-PCR and semi-quantitative RT-PCR

DNase treatment is necessary for every method.

1)　 RNeasy (QIAGEN)

Follow manufacturer's manual.

Note:

- This method is as easy as the method 2).

- Avoid overloading sample onto column.

- genomic DNA usually contaminate with RNA.

- 100 µg total RNA from 0.2 g fresh weight of 2-cell stages of protonema regenerated from protoplasts.

- RLT buffer in the kit works well for protonemata.

2)　 ISOGEN, ISOGEN-LS (NIPPON GENE, http://nippongene.com/)

Follow manufacturer's manual.

Note:

- This method is as easy as the method 1).　 However purity of total RNA from gametophore is poor and it is hard to concentrate total RNA more than 200ng/µl.

- This kit is used to get total RNA without genomic DNA.　 In some cases, we extract RNA with RNeasy kit, then use ISOGEN to remove genomic DNA contaminants from the RNA fraction.

- We extracted total RNA with this kit from young sporophytes with archegonium (~30 µg from 14,500 archegonium, T. Nishiyama, unpublished).

- For protonemata: 50 µg/g fresh weight OD260/OD280 = 1.8

and for gametophore: 10 µg/g fresh weight OD260/OD280 = 1.2 (yellowish color)

- Remove gel-like precipitation (when for protonema) or soft precipitation (gametophore) by pipetting not to touch RNA pellet, when LiCl precipitation is carried out.　 When EtOH precipitation is performed, translucent pellet contains RNA.

3)　 GuSCN and CTAB method

<Extraction of total RNA>　 (large scale)　 (It takes ~8 h)

1. Buffer GnSCN　　　　　　　　　　　 final conc.　　　　 15ml　　 30ml　　 50ml　　

GuSCN (Guanidine thiocyanate mw=118.2)

4 M　　　　　　　　 7.1 g　　 14.2 g　 24 g

NH4SCN (ammonium thiocyanate mw=76.12)

1 M　　　　　　　　 1.14 g　 2.28 g　 3.8 g

1 M Tris-HCl (pH 7.5)　　　　　　　 100 mM　　　　　　　　　　　 1.5 ml　 3 ml　　 5 ml

N-lauryl sarcosine　　　　　　　　　　 1%　　　　　　　　　 0.15 g　 0.3 g　　 0.5 g

Antifoam A Emulsion (sigma A5758)trace　　　　　　　　　　　 1 drop　 1 drop　 1drop

PVP 360,000　　　　　　　　　　　　　　 < 0.5%　　　　　　 0.075 g 0.15 g　 0.25 g

beta-mercaptoethanol　　　　　　　　 1%　　　　　　　　　 150 µl　 300 µl　 500 µl

2. CTAB buffer　　　　　　　　　　　　 final conc.　　　　 50 ml　 100 ml

CTAB　　　　　　　　　　　　　　　　　　　 2%　　　　　　　　　 1 g　　　 2 g

1 M Tris.HCl , pH7.5　　　　　　　　 50 mM　　　　　　 2.5 ml　 5 ml

0.5 M EDTA.Na2　　　　　　　　　　　 5 mM　　　　　　　 0.5 ml　 1 ml　　

5 M NaCl　　　　　　　　　　　　　　　　 0.84 M　　　　　　 8.4 ml　 16.8 ml

up to 　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　 50 ml　 100 ml

Add beta-mercaptoethanol just before use　　　　　　 500 µl　 1 ml

3. 10% CTAB buffer　　　　　　　　 final conc.　　　　 50 ml

CTAB　　　　　　　　　　　　　　　　　　　 10%　　　　　　　　 5 g　　　

5M NaCl　　　　　　　　　　　　　　　　　 0.7 M　　　　　　　 7 ml　　　　　　　　

DEPC water up to 50 ml

4.CTAB precipitation buffer　　　 final conc.　　　　 50 ml

CTAB　　　　　　　　　　　　　　　　　　　 1%　　　　　　　　　 0.5 g

1 M Tris.HCl, pH 7-8　　　　　　　　 50 mM　　　　　　 2.5 ml

0.5 M EDTA　　　　　　　　　　　　　　 5 mM　　　　　　　 500 ul

DEPC water　 up to　　　　　　　　　　　　　　　　　　　　　 50 ml

5. High-salt TE　　　　　　　　　　　　　　　　　　

6. Chloroform/Isoamylalcohol = 24 : 1

0.5 ml Tris.HCl, pH 7-8

0.1 ml 0.5 M EDTA

10 ml 5 M NaCl (final 1M)

DEPC water to 50 ml

Procedure

1.　　　　 Grind in liqN2 with mortar and pestle

2.　　　　 Transfer powder to 50 ml tube with 15 ml GuSCN B　 (5 g moss/15 ml at most)

3.　　　　 Shake vigorously

4.　　　　 Add 15 ml of C/I　 (seal with parafilm)

5.　　　　 Shake vigorously for 5 min.

6.　　　　 Cfg at 6K rpm (Hitachi himac CR20E, R12A2 rotor, #25) for 12 min. @RT

7.　　　　 Collect aqueous phase　　 (should be a little brownish)

8.　　　　 Repeat C/I extraction two more　 (total 3 times)

9.　　　　 Collect aqueous phase (ca. 14 ml)

10.　　　 Add 1/10 vol of 3 M NaOAc (pH 5.2) and 2-2.5 vol of 100% of EtOH　 (become cloudy)

11.　　　 Place on ice for 30 to 60 min.

12.　　　 Cfg at 9K rpm for 20 min. at 4˚C

13.　　　 Rinse pellet with 80% EtOH, once

14.　　　 Resuspend in 5 ml of CTAB B　 (combine tubes, 15-20 ml/tube, ideally 15 ml)*

(warm at 55˚C for 20 min,　 pipetting to <1mm pieces, then cool to RT)

(*)　 you may use falcon2059 and cfg 5k-6k (or 7k with adaptor) with angle roter #7

15.　　　 Add 5 ml of C/I and vortex well

16.　　　 Cfg at 5K for 10 min. at RT　　　 (thin white interphase)

17.　　　 Harvest the sup (~ 5 ml)

18.　　　 Add 0.6 ml of 10% CTAB and mix well

19.　　　 Add 5.6 ml (1 vol) of C/I, then mix

20.　　　 Cfg at 5K for 20 min. at RT

21.　　　 Take sup　 (sup= about 5 ml,　 almost no interphase)

22.　　　 Add 2-ME to 0.5%

　　　　　 Add 1.2 vol (6ml) of CTAB pptn B　　　　 (become cloudy)

23.　　　 Incubate at RT for 30 to 60 min.

24.　　　 Cfg at 9K for 15 min at RT

25.　　　 Dissolve the pptn in 5 ml High-Salt TE B (add 2-ME at 0.5% just before use),

then heat to 50-60 ˚C for 2 – 3 min　 (by pipetting)

26.　　　 Add 2.5 vol (12.5 ml) of 100% EtOH

27.　　　 Place on ice for 30 to 60 min.

28.　　　 Cfg at 9K for 20 min. at 4˚C

29.　　　 Rinse with 80% EtOH once

30.　　　 Dissolve in 300 µl TE (300 µl/ a 50 ml tube)

This resultant fraction contains total RNA and genomic DNA.　 To remove genomic DNA, we usually perform the following protocol by using ISOGEN-LS (NIPPON GENE).　

(300 µl; convenient for ISOGEN-LS, see below)

(by pipetting and still cloudy)

<ISOGEN-LS (NIPPON GENE)>　 (2.5 h)

31.　　　 Transfer the solution into 1.5 ml tube　 (<300 µl / a tube)

32.　　　 Add 3 vol. of ISOGEN-LS (900 ul /tube)

33.　　　 Mix well and incubate for 5 min at RT

34.　　　 Add 0.8 vol. (300*0.8=240 µl) of CHCl3 (-IAA)

35.　　　 Shake vigorously for 15 sec and then incubate for 2 to 3 min. at RT

36.　　　 Cfg at 15K rpm for 15 min. at 4ºC

37.　　　 Collect aqueous phase　 (about a half of the above sum, ~750 ul)

38.　　　 Add 1 vol. of isopropanol

39.　　　 Incubate for 10 min at RT

40.　　　 Cfg at 15K for 10 min at 4˚C

41.　　　 Rinse with 80% EtOH, once

42.　　　 Dry

43.　　　 Dissolve in H2O or TE　 (~150 µl / 1.5 ml tube = 50 ml tube, if 5 g, 200 µg RNA/150 µl)

44.　　　 AGE to check　　 (loading 1ul is usually enough even in DNA gel)

45.　　　 Quantification by spectrophotometer　　
4) DYNABEADS mRNA DIRECT kit (DYNAL)

Meterials

・ 3 g fresh or frozen samples

Reagents

DYNABEADS mRNA DIRECT kit (DYNAL) or prepare the following reagents

　

・Dynabeads Oligo (dT)25 (DYNAL) Magnetic beads

・ Lysis/Bindingbuffer

100 mM Tris-HCl pH 8.0

500 mM LiCl

10 mM EDTA pH 8.0

1% SDS

5 mM DTT

・ SDS+Washingbuffer

10 mM Tris-HCl pH 8.0

0.15 M LiCl　　

1 mM EDTA

0.1% SDS

・ Washingbuffer

10 mM Tris-HCl pH 8.0

0.15 M LiCl　　

1 mM EDTA

・ Elutionbuffer

2 mM EDTA pH 8.0

Magnetic stand for recover magnetic beads

・ Dynal MPC-1

・ Dynal MPC-E-1

・ Heat block

Protocol

Follow the manufacturer's instruction, protocol B-for large scale mRNA isolation

Note

As the purity of poly(A)+ RNA obtained in this way is not good, we further purify the RNA by ISOGEN-LS (NIPPON GENE).

Yield and purity just after the extraction with

protonemata: 3 ug poly(A)+RNA/g fresh weight,　 OD260/OD280=1.6

gametophores: 1-2 ug poly(A)+RNA/g fresh weight,　 OD260/OD280=1.6