| 1/8 | extract_protocol_id: | 3 | | extract_protocol_name: | Hot Phenol/Guanidine Thiocyanate and RNeasy Clean up | | amplification: | none | | extract_protocol: | TRIzol-like RNA isolation and RNeasy clean up protocols
TRIzol-like Reagent: Final Concentration
Phenol, pH. 4.3 380 ml 38%
Guanidine thiocyanate 118.6 g 1 M
Ammonium thiocyanate 76.12 g 1 M
3M Sodium Acetate, pH 5.0 33.4 0.1 M
Glycerol 50 ml
H2O to 1 liter
Total 1 liter
**This reagent works better than commercial TRIzol and is significantly cheaper if you are preparing lots of RNA samples**
0.8 M Sodium citrate/1.2 M NaCl
Isopropanol
Chloroform
75% ETOH
DEPC-H2O
Work in the hood. Wear safety glasses when working with TRIzol and chloroform.
Preheat the Trizol to 60oC for at least 1 hr.
1. Grind 1-gram tissue with a mortar and pestle in liquid nitrogen.
2. Add 10 ml TRIzol-like reagent (pre-heated to 60oC) to tissue in a falcon tube. Swirl to mix.
3. Incubate samples at 60oC for 5 minutes.
4. Centrifuge at 9500 x rpm at 4°C for 10 minutes. Transfer supernatant to 50 ml polypropylene copolymer tube (Nalgene Round Non-lipped Centrifuge tube #3110-0500) centrifuge tube that has been dipped in chloroform to rinse out any residual RNases.
5. Add 2 ml of chloroform (Fisher #C298-4). Vortex the sample until color shade is uniform, at least 5 seconds, and incubate at room temperature for 5 minutes.
6. Centrifuge @ 9000 x rpm for 15 minutes at 4°C.
7. Collect the upper aqueous layer and transfer to a new 50 ml pp (SS34) centrifuge tube dipped in chloroform.
8. Add 0.5 volume of isopropanol & Na-citrate/NaCl solution per 1 ml of aqueous solution. Mix gently. Incubate at room temperature for 20 minutes.
9. Centrifuge at 9000 rpm for 10 minutes at 4°C.
10. Wash the clear RNA pellet with 10 ml of cold 75% ethanol. Swirl & centrifuge at 9000 rpm for 5 minutes at 4°C.
11. Discard supernatant & air-dry the pellet for 10 minutes, inverted on a kimwipe. Dissolve pellet in 300 ul of AccuGENE Molecular Biology water (Cambrex #51200). Resuspend by pipeting up & down a few times.
12. Add 2 ul Recombinant RNasin Ribonuclease Inhibitor (Promega #N2515). Incubate at 55-60°C for 10 minutes to resuspend.
13. Transfer to 1.5 ml microcentrifuge tube and rinse the 50 ml pp (SS34) centrifuge tube with 100 ul DEPC-treated H2O. Check concentration and run a gel of 2 ul of RNA samples (see qualitative/quantitative analysis). At this stage we have very clean RNA at a 260/280 of ~1.9 –> 2.1, however for consistent labeling for GeneChip hybridizations, proceed to step #15.
RNeasy Clean-up (Midi Kit)
Mercaptoethanol must be added to Buffer RLT (10ul/1ml RLT).
15. Adjust sample to 0.5 ml volume with RNase-free water and add 2.0 ml Buffer RLT. Mix thoroughly.
16. Add 1.4 ml (96-100%) ethanol to the diluted RNA. Mix thoroughly.
17. Apply sample to a Midi column placed in a 15 ml centrifuge tube. Maximum loading volume is 4.0 ml. Close tube gently and centrifuge for 5 min at 4750 rpm. Discard the flow-through.
18. Add 2.5 ml Buffer RPE to the RNA easy column. Close the centrifuge tube gently, incubate for 3 min and centrifuge for 2 min at 4750 rpm. Discard the flow-through.
19. Add another 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube gently, incubate for 3 min and centrifuge for 5 min at 4750 rpm to dry the RNeasy silica-gel membrane. Remove flow-through and centrifuge again for another 3 min.
20. Transfer the RNA easy column to a new collection tube. Pipet 250 ml volume of RNase-free water directly onto the RNeasy silica-membrane. Close the tube gently. Let it stand for 1 min and then centrifuge for 4 min at 4750 rpm. Do a double elution for better recovery (Put the eluate back to the column and centrifuge again. We usually get a 50% recovery with double elution).
Qualitative/Quantitative Analysis
1. To determine concentration, dilute 2 ul RNA to 78 ul H20. Measure the absorbancy 260/280 and calculate concentration.
2. Prepare and run a 1% gel (2 ul of RNA, 7 ul H2O and 1 ul of tracking dye).
- use 1X TBE-agarose
- run gel at 80 volts for 1.5 hrs | | remark: | | | last_update: | 2003-09-19 10:55:29 | | extract_na: | total RNA | | login_id: | caldo | | resource: | Affymetrix manual |
| 2/8 | extract_protocol_id: | 9 | | extract_protocol_name: | NASC total RNA extraction | | amplification: | T7 RNA Polymerase | | extract_protocol: | Extraction Protocol Information
Name of Protocol: Logeman et al,
Method:
Protocol As in Logemann et al. 1987
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
| | remark: | | | last_update: | 2004-02-24 19:49:39 | | extract_na: | total RNA | | login_id: | da_xia | | resource: | |
| 3/8 | extract_protocol_id: | 7 | | extract_protocol_name: | RNA isolation from starchy endosperm | | amplification: | | | extract_protocol: | RNA extraction Protocol for Seed Tissue (and leaf tissue, however the pre-extraction with the buffer solution prior to Trizol is not necessary for leaves. If you go right to Trizol with seed tissue, your RNA "pellet" will be almost entirely starch. Note: Of course gloves should be worn throughout this procedure. Also, samples should be on ice and all solutions used should be relatively sterile.)
1. Grind sample in liquid Nitrogen. Keep powder frozen. If your sample is not ground to the consistency of flour, your yields will likely be lowered.
2. Add enough powder to a 2 ml tube to equal roughly 0.2 to 0.5 cc of volume.
(Note: if you use much more powder than this, you will not recover all of the RNA, nor will your RNA be very intact or clean.)
3.Add 0.5 ml Extraction Buffer and vortex. (Note: you will need to let your frozen samples warm up slightly before adding this buffer. Of course, if your samples freeze into a cube, they should be thawed and then vortexed again till homogeneous.)
4.Add 0.5 ml Phenol/Chloroform/Isoamyl alcohol (49:49:2) and vortex till thoroughly suspended.
5.Spin 5 minutes at 14,000 RPM in large Sorvall centrifuge at 4 C.
6.Remove 0.5 ml of upper aqueous phase to a fresh 2 ml tube being careful to avoid the interphase material.
7.Add 1.0 ml Trizol reagent and vortex (your sample at this point will be completely incorporated into the Trizol solution and should be invisible).
8.Add 0.2 ml Chloroform and vortex and the phases will separate just like magic.
9.Spin 14,000 for five minutes at 4 C.
10.Remove 0.75 ml of upper aqueous to a fresh 2 ml tube. (This is not all of the upper phase. However, if you contaminate your sample with the interphase material your RNA will be of lower quality and hard to accurately quantify.)
11.Add 0.5 ml Chloroform and vortex.
12.Spin 14,000 for five minutes at 4 C.
13.Remove 0.6 ml of upper aqueous to a fresh 2 ml tube.
14.Add 1.2 ml of 100% ethanol and 60 ul of 3 M Sodium Acetate.
15.Vortex and incubate at -80 C for > 1 hr.
16.Spin 14,000 for 20 minutes at 4 C.
17.Decant supernatants, and wash pellets with 70% ethanol, and briefly respin. Decant or aspirate most of remaining ethanol. Do not completely dry your RNA as it will then be quite difficult to resuspend in TE.
18.Resuspend RNA pellets in 50 ul of sterile TE by vortexing. Expected yield is 25 to 100 ug.
19. Assay 2 ul in 200 ul TE on the spectrophotometer.. This should give readings in the 0.1-0.3 range. To examine degree of intactness of your RNA, load 1 ug of each sample on a standard agarose gel and photograph. You should see the two Ribosomal bands and very little if any smearing forward of the ribosomal bands which would indicate degradation. Note: your A260/A280 ratios should be greater than 1.8 with this procedure. They will likely be 2.0 +/- 0.1.
Extraction Buffer (make up fresh day of use.) 20 mls.
50 mM Tris pH 9.0 1 ml 1 M Tris pH 9.0
200 mM NaCl 2 ml 2 M NaCl
1% Sarcosyl 2 ml 10% Sarcosyl
20mM EDTA 0.8 ml 0.5 M EDTA
5 mM DTT 0.1 ml 1M DTT (freezer aliquots.)
H2O 14.1
| | remark: | The protocol comes from Mike Giroux, mgiroux@montana.edu
| | last_update: | 2003-10-09 07:27:56 | | extract_na: | total RNA | | login_id: | arnis | | resource: | |
| 4/8 | extract_protocol_id: | 10 | | extract_protocol_name: | RNeasy Midi + PEG | | amplification: | none | | extract_protocol: | The RNeasy Midi (Qiagen, Valencia, CA) + PEG method was performed according to the manufacturer’s instructions for isolation of RNA from plants, with a few modifications; 3.3% (w/v) PEG (MW 15,000-20,000) was added per mL of buffer RLT prior to the addition of the plant material (Gehrig et al. 2000). Following initial centrifugation, the supernatant was filtered through Miracloth. Final elution was done using 150 µL RNase-free water, and the eluate was run back through the column for the second elution. RNeasy cleanup was performed as described by the manufacturer, except that no more than 70 μg RNA was applied to the column. | | remark: | | | last_update: | 2005-07-11 12:40:59 | | extract_na: | total RNA | | login_id: | elizART | | resource: | |
| 5/8 | extract_protocol_id: | 11 | | extract_protocol_name: | Tris-LiCl | | amplification: | none | | extract_protocol: | A Tris-LiCl method (Wang et al. 2000) was modified by adding a phenol:chloroform extraction step. In short, the extraction buffer consisted of 200 mM Tris-HCl pH 8.5, 1.5% (w/v) lithium dodecyl sulfate, 300 mM Na-EDTA (ethylenediaminetetraacetic acid), 1% (w/v) sodium deoxycholate, 1% (v/v) tergitol NP-40. Just before use, 2 mM ATA, 20 mM DTT, 10 mM thiourea and 2% (w/v) PVPP (polyvinylpolypyrrolidone) were added. Approximately 1.5 g leaf or 3 g berry or root tissue was ground in a liquid nitrogen cooled mortar, and combined with 25 mL extraction buffer. The mixture was frozen at –80°C for at least 2 hr. The homogenate was thawed in a 37°C water bath, and centrifuged for 20 min at 4°C and 5,000 x g. The supernatant was transferred to fresh tubes, and 0.106 M sodium acetate and 10% (v/v) ethanol added. After 10 min on ice, this was centrifuged 4°C for 20 min at 5,000 x g. The supernatant was retained and 0.33 M sodium acetate and 33% (v/v) isopropanol was added. The mixture was incubated at least 2 hr at -20°C. The tubes were then centrifuged for 30 min at 4°C and 5,000 x g. The pellet was resuspended in 3 mL TE (10 mM Tris pH 7.5, 1 mM EDTA), and incubated on ice 30 min, then centrifuged for 30 min at 4°C and 5,000 x g. The supernatant was transferred to fresh tubes, LiCl added to 2.5 M, and incubated on ice at 4°C overnight. The tubes were centrifuged for 30 min at 4°C and 10,000 x g. The pellet was resuspended in 1.5 mL TE and potassium acetate was added to 3 M. The mixture was incubated on ice for 3 hr, and centrifuged for 30 min at 4°C and 10,000 x g. The pellet was resuspended in 1 mL TE, and a single phenol:chloroform:isoamyl alcohol (25:24:1) extraction performed, followed by a single chloroform:isoamyl alcohol (24:1) extraction. Ten percent 3.3 M sodium acetate and 2 volumes ethanol were added and incubated at –20°C for at least 2 hr. Tubes were centrifuged at 4°C and 10,000 x g for 30 min. The pellets were washed with 500 µL absolute ethanol, dried on ice, and resuspended in 500 µL DEPC-treated water. RNeasy cleanup was performed as described by the manufacturer, except that no more than 70 μg RNA was applied to the column. | | remark: | | | last_update: | 2005-08-09 12:59:04 | | extract_na: | total RNA | | login_id: | elizART | | resource: | |
| 6/8 | extract_protocol_id: | 12 | | extract_protocol_name: | Modified Pine Buffer RNA Extraction Procedure | | amplification: | | | extract_protocol: | The total RNA extraction procedure of Chang et al 1993 was used with a few modifications. The pellet was resuspended in TE, DNAsed and extracted with a single phenol:chloroform:isoamyl alcohol (25:24:1) followed by two single chloroform:isoamyl alcohol (24:1) extractions. RNeasy cleanup was performed as described by the manufacturer.
A simple and efficient method for isolating RNA from pine trees. Chang, S; Puryear, J; Cairney, J, Plant molecular biology reporter - ISPMB. June 1993. v. 11 (2) p. 113-116 | | remark: | | | last_update: | 2005-09-21 15:42:23 | | extract_na: | | | login_id: | fennella8 | | resource: | |
| 7/8 | extract_protocol_id: | 13 | | extract_protocol_name: | RNA Isolation Using Trizol And Qiagen RNAeasy Columns | | amplification: | none | | extract_protocol: | http://www.maizearray.org/maize_protocols.shtml
Total RNA Isolation Using Trizol and Qiagen RNAeasy Columns
Overview
This protocol is quick and works very well for preparing 20 to 40 ug of very clean, salt free,
RNA. The RNA prepared from this protocol is ready for target preparation using the Ambion
Message Amp II procedure to produce aminoallyl labeled cRNA.
Materials Required
RNAase-free mortar and pestle: cover the mortar and pestle with aluminum foil and bake
at least 3 hours at 180°C
RNAase-free 1.5 or 2.0 mL (preferred) microfuge tubes
Liquid nitrogen
Microfuge
RNAase-free pipette tips
Qiagen RNAeasy Mini elute columns and buffers (Qiagen Cat # 74204)
DEPC-treated H2O
Trizol (Invitrogen)
Procedure
1. Homogenize tissue in liquid nitrogen. It is not necessary to homogenize large amounts of
tissue as the Qiagen RNAeasy columns can only bind ~40 ug of RNA and overloading
the column is not advised. If you are working with pooled samples you may find that you
have more ground sample than you can use. It is convenient to have a small measuring
device (teaspoon, grooved spatula, etc) to transfer the ground material directly to the
Trizol in the microfuge tube.
a. Chill mortar with ~100 mL of liquid nitrogen.
b. Add frozen tissue after nitrogen is nearly completely evaporated.
c. Grind tissue quickly but carefully.
d. When liquid has fully evaporated, grind faster to produce a fine talc-like powder.
2. Add 1/8 to 1/4 teaspoon to 1.0 ml of Trizol. It is important to mix well immediately by
vortexing and not allow tissue to thaw that is not in contact with the Trizol. You may
want to prewarm the Trizol (35-40 C) so that it does not freeze when it comes into
contact with the frozen tissue. This is the most critical step in the procedure as once the
tissue is completely mixed with the Trizol, it is protected from RNAases.
3. Incubate for five minutes at room temperature (RT), votexing frequently.
4. Add 0.2 ml of chloroform to the Trizol, and vortex for 15 seconds.
5. Incubate for 1 minute at RT, vortex again for 15 seconds.
6. Centrifuge at 15,000 xg for 10 minutes to separate phases
7. Remove 200 ul from the top layer and add to 700 ul of Qiagen RLT buffer in a new tube.
Remove the rest of the top layer and freeze at -20 to serve as a backup in case your initial
yield is low.
8. To the 200 ul of sample now combined with 700 ul RLT buffer, add 500 ul of 96-100%
ethanol. Mix well by vortexing but do not centrifuge.
1
9. Apply half of your sample (~700 ul) to a Qiagen MinElute spin column placed in a 2 ml
microfuge tube. Spin 15 seconds at ~10,000 rpm. Discard flow through and repeat
procedure with the second half of your sample.
10. Remove the Minelute column to a new 2 ml microfuge tube and add 500 ul of RPE to the
spin column. Spin 15 seconds at ~10,000 RPM. Discard flow through.
11. Add 750 ul of 80% ethanol and spin at ~10,000 rpm for 15 seconds. Repeat this step a
second time with another 750 ul of 80% ethanol. This step is repeated to ensure removal
of all guanidine salts that may inhibit downstream applications.
12. Transfer the Minelute spin column to a new 2 ml microfuge tube. Spin for 5 minutes at
top speed with the cap off. This ensures the removal of trace amounts of ethanol that
may interfere with downstream applications.
13. To elute RNA, transfer spin column to a new, 1.5 ml microfuge tube. Elute with 10 ul of
RNAase free water. Repeat with another 10 ul of RNAase free water. If you suspect low
RNA concentration, you may elute with 12 ul of RNAase free water. It is desirable to
have a concentration of ~ 1 ug/ul if possible. Check concentration on a gel or
spectrophotometer. If your initial yield from the 200 ul is low, you may want to consider
precipitating the remainder of your sample with an equal volume of isopropanol and
resuspending in 200 ul of H2O for concentration using a minelute RNAeasy column.
| | remark: | This is the protocol recommended by NSF maizearray project. | | last_update: | 2005-10-04 07:49:00 | | extract_na: | total RNA | | login_id: | da_xia | | resource: | |
| 8/8 | extract_protocol_id: | 14 | | extract_protocol_name: | RNA Prep. for Kernels | | amplification: | none | | extract_protocol: | 1. Grind 2.0 gm frozen kernels in pre-chilled coffee mill (chilled with dry ice) until fine powder. Scrape the powder into a pre-chilled mortor (chilled with liquid nitrogen). Grind with pestle to a fine, homogeneous powder.
2. Scrape powder into a 100 ml beaker containing 6.0 ml lysis buffer (0.1 M NaCl, 50mM Tris-HCl, pH 7.4, 50 mM EDTA, 2% SDS, plus 200 ug/ml proteinase K added just before use). Stir with magnetic stirrer at RT, 5 min.
3. Add 6.0 ml phenol (pH 8.0):chloroform:isoamyl alcohol (25:24:1). Stir 10 min. RT.
4. Transfer to 15 ml plastic conical centrifuge tubes and spin 5K rpm for 10. min. at RT in a swinging bucket rotor.
5. Re-extract supernatant with 6.0 ml phenol: chlor.:IAA. Stir 10 min. at RT. Spin as above.
6. Measure volume of aqueous phase and transfer to a baked 30 ml Corex tube on ice. Add an equal vol. of 4 M LiCl. Mix well and leave on ice in the cold room O/N.
7. Spin 10K rpm, 10 min., 4 degrees C. in swinging bucket rotor.
8. Resuspend pellet in 0.5 ml DEPC-treated water. Add 50 ul 3M NaOAc and 1.25 ml EtOH.
9. Chill at -20 degrees about 1 hr.
10. Spin 10K rpm, 4 degrees in swinging bucket rotor.
11. Resuspend pellet in 0.45 ml DEPC-treated water. Dilute 5 ul in 495 ul water and read A260. | | remark: | | | last_update: | 2005-10-07 15:54:43 | | extract_na: | total RNA | | login_id: | HakeOligo | | resource: | |
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