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Please direct all comments, questions, and suggestions to biobzc@hofstra.edu
PREPARATION OF TOTAL AND POLY A+ RNA /NORTHERN ANALYSIS
You will prepare Poly A+ (messenger) RNA from whole adult flies to be used for a Northern Analysis. To obtain Poly A+ RNA you will first isolate total RNA. On average 1 µg of total RNA can be extract from one adult fruit fly. Poly A+ RNA is 2% of total RNA.
Total RNA Isolation
WHEN WORKING WITH RNA ALWAYS WEAR GLOVES, MAKE UP SOLUTIONS IN DEPC-TREATED WATER, USE ONLY RNA GLASSWARE AND PLASTIC SUPPLIES, & KEEP RNA ON ICE AT ALL TIMES
Solutions for Drosophila RNA preparation
SOLUTION 1: 6 M Urea, 3 M LiCl (Don’t autoclave and use fresh, 1-2 weeks max.)
SOLUTION 2: 10 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS (Autoclave before use)
DAY 1
1. Homogenize ~500 flies/larvae or 500 µl (1.5 microcentrifuge tube) of embryos in 4-5 ml of Solution 1.
2. Transfer the homogenate to 4 microcentrifuge tubes. Rinse the homogenizer with an additional 1 ml of Solution 1 and add to initial homogenate.
3. Place in an RNase free area at 0oC overnight (in ice bucket in the refrigerator).
DAY 2
1. Centrifuge homogenates for 10 min at 4oC at 14,000 RPM.
2. Remove supernatant and discard. Re-suspend pellet in 500 µl of Solution 2; vortex to re-suspend.
3. Add 500 µl of phenol saturated with depc water pH 5.2 and vortex for 15 seconds.
4. Add 500 µl of chloroform and vortex as above.
5. Centrifuge at room temperature for 5 min at 14K RPM.
6. Transfer aqueous layer (top) to a new tube and discard the bottom layer.
7. Repeat steps 3-6 three more times (until aqueous layer is clear).
8. Add 500 µl of chloroform and centrifuge as above. Remove aqueous layer as above and add 1/10th volume of 3 M sodium acetate, pH 5.2 and 2.5 volumes cold 100% EtOH
9. Incubate in an RNAse free area at -20oC overnight.
DAY 3
1. Centrifuge RNA preparations at 14K RPM at 4oC for 15 min.
2. Remove & discard supernatant. Wash pellets with 200 µl of cold 70% EtOH.
3. Centrifuge at 14 K RPM, 4oC for 5 minutes.
4. CAREFULLY remove supernatant (pellets are very loose now).
5. Let air dry 10-20 minutes.
6. Combine samples and re-suspend in a total of 500µl DEPC-treated water
7. Run a test gel of 1-2 µl of Total RNA.
8. Store in an RNAse free area at -20oC or at -80oC for long term use.
Poly A+ Isolation
| Loading Buffer | Middle Wash Buffer |
| 0.5M LiCl | 0.15M LiCl |
| 10mM Tris-HCl, pH 7.5 | 10mM Tris-HCl, pH 7.5 |
| 1mM EDTA, pH 8.0 | 1mM EDTA, pH 8.0 |
| 0.1% SDS | 0.1% SDS |
DAY 1
1. Add 20 mg oligo dT cellulose to a 1.5 ml microcentrifuge tube, mix in 0.5ml of 0.1N NaOH, 5.0mM EDTA to activate. Spin the column briefly and remove supernatant.
2. Wash the column 3X with DEPC-treated water.
3. Wash the column with 1.0 ml loading buffer. Re-suspend the washed column in 0.2 ml loading buffer (column must be used within 1 hour).
4. Heat the total RNA sample at 65-70oC for 10 minutes.
5. Calculate the volume of 10M LiCl you would need to add to the RNA sample to make the LiCl concentration 0.5M in the total mixture. Add this volume to the RNA.
6. Add the RNA plus LiCl to the prepared column, cap tube tightly, seal with parafilm and rock GENTLY for about 1 hour at room temperature.
7. Centrifuge the column at 3000 RPM for 10 seconds and remove supernatant. IT IS IMPORTANT TO CENTRIFUGE AT 3000 RPM OR LOWER TO AVOID STRIPPING THE mRNA FROM THE COLUMN.
8. Add 0.3 ml loading buffer, mix, and centrifuge as above, remove supernatant. Do this three times. The supernatant should be clear at this point, if not keep washing as above.
9. Re-suspend the column material in 0.5 ml loading buffer.
10. Add the column material to the top chamber of an RNase-free microcentrifuge spin column. Make sure to GET ALL OF THE CELLULOSE INTO THE SPIN COLUMN.
11. Centrifuge at 3000 RPM for 10 seconds and discard the fluid (in bottom chamber).
12. Wash the column material with 0.3 ml middle wash buffer and centrifuge as above. Repeat this wash a total of 3X (DO NOT VORTEX OR INVERT TUBES AND MAKE SURE NOT TO PUNCTURE THE SPIN COLUMN MEMBRANE WITH PIPETTE TIP).
13. Transfer the top chamber of the spin column to a new lower chamber.
14. Elute the Poly A+ RNA by adding 0.2 ml DEPC-treated water to the column material; mix gently with pipette tip and centrifuge as above. SAVE THE FLOW THROUGH!!! Repeat this once (0.4ml DEPC-treated water total). If some cellulose has been spun through the column, spin the bottom section only at 14,000 RPM for ~1 minute, and transfer supernatant to a new tube.
15. Add 2.5 volumes (1ml) of cold 100% EtOH and 1/10th volume (40ml) 3 M NaOAc, pH 5.2.
16. Place an RNAse free area at -20oC overnight.
DAY 2
1. Centrifuge the Poly A+ preparation at 14,000 RPM at 4oC for 10 min.
2. Remove the supernatant. Wash pellets with 200 ml of cold 70% EtOH. You probably will not see the pellet at this point but have faith.
3. Centrifuge again as in step 1.
4. CAREFULLY remove supernatant (pellets may be very loose now).
5. Let air dry 10-20 minutes.
6. Re-suspend pellets in 5-10 ml of DEPC-treated water.
7. Store in an RNAse free area at -20oC or -80oC for long term use.
8. For use on a Poly A+ Blot, you should load 8-10 µg RNA in a lane. Poly A+ is typically 2% of Total RNA. If you start with 500 flies, 500 x 2%=10µg mRNA if your recovery is 100%.
Denaturing Agarose Gel of Poly A+ RNA
RNA is always denatured by heating before gel electrophoresis and it is fractionated on a denaturing gel to minimize RNA secondary structure formation.
Preparation of the Gel
An RNA gel consists of 1.5% agarose in 1X MOPS buffer/16-17 % formaldehyde. Since MOPS is heat sensitive and formaldehyde should not be heated, the agarose is dissolved in DEPC-treated water and then equilibrated to 50oC. The MOPS and formaldehyde are added quickly immediately before the gel is poured.
Preparation of the Gel Apparatus
Unless you have an RNA-dedicated gel apparatus, the gel box and comb should be soaked in 1% SDS in DEPC-treated water for 15 minutes and rinsed with DEPC-treated water before use.
Running Buffer
1X MOPS, 16-17% formaldehyde in DEPC-treated water
Preparation of RNA
Add the desired amount of RNA to RNA loading buffer (see below), incubate at 65-70oC for 10 minutes. Add an appropriate amount of standard loading buffer and load the samples in the gel immediately. If there is a delay between heating and loading of the samples hold them on ice.
RNA Loading Buffer
50% deionized formamide (formamide in freshly-opened bottles is deionized, it is kept de-ionized by freezing. Thaw frozen formamide on ice — do not heat to thaw.)
16-17% formaldehyde
1X MOPS
DEPC-treated water to volume
0.2 µl ethidium bromide
Make a Master Mix of the RNA Loading Buffer and then aliquot it to the RNA samples. This will ensure that you have the same amounts of each ingredient in each sample. I usually make up one extra aliquot to compensate for the accumulation of small pipeting errors. When calculating the number of samples do not forget to include the RNA ladder, it must be treated as if it were a sample.
Example: Preparation of RNA plus loading buffer to run 2 samples plus ladder.
|
Ingredient |
Amount/10µl |
# of Samples + 1 |
Master Mix |
|
RNA |
2 |
||
|
formamide |
5 |
4 |
20 |
|
formaldehyde |
1.6 |
" |
6.4 |
|
MOPS |
1 |
" |
4 |
|
DEPC-treated water |
0.4 |
1.6 |
|
|
Ethidium bromide |
" |
.5 |
Add 8 µl of the Master Mix to each 2 µl RNA sample, heat, then add 1 µl of standard loading buffer and load the sample into the gel.
Northern Blot - see Genomic Southern Analysis, Southern blot