Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The overall goal of this procedure is to allow the detection of small RNA expression from Deano gaster embryos or tissues or cells. This is accomplished by first extracting RNA from the selected biological sources. The second step is to fractionate RNA by electrophoresis along an acrylamide gel to resolve low molecular weight migration patterns.

Next, the RNA species are transferred and chemically cross-linked to a neutral solid support. The final step is to label candidate molecules by annealing them with specific antisense probes. Ultimately, auto radiography is used to reveal signals that reflect the expression of molecules under analysis.

This unanswered north block method cannot answer key questions in the RNA biology field, such as the steady state levels of fully expressed RNA molecules. To begin grow drosophila Schneider's two cells to a density of one to five times 10 to the sixth per milliliter. Then detach the semi adherent cells by pipetting and centrifuge the cells at 100 GS per one minute.

Alternatively, accumulate embryos on egg laying plates. Wipe embryos off with a paintbrush and distilled water, filter them through a double sieve, then rinse and recover the embryos in a tube, add 100 microliters of RNA extraction, reagent and pestle. Homogenize the sample.

Perform RNA isolation following the manufacturer's instructions. Next, assess the RNA concentration by a UV spectrophotometer and ensure the RNA purity is close to 2.0 based on an A two 60. Over a two 80 reading.

Then prepare a denaturing aros gel and allow the gel solution to cool down. When the temperature reaches 60 degrees Celsius, add 10 milliliters of 10 x mops buffer, and eight milliliters of 37%formaldehyde. Pour the gel into a horizontal electrophoresis cell.

Let the gel solidify under a hood for one hour following solidification, submerge the gel into 500 milliliters of one x mops buffer. Next Eloqua, two micrograms of RNA sample and one microgram of ladder. Add three volumes of aros loading dye to the RNA and to the ladder.

Heat the samples at 80 degrees Celsius for five minutes and immediately cool them on ice. Load the samples and run the gel at 50 volts for one hour with occasional buffer recycling. Prepare 10 milliliters of 10%acrylamide bis acrylamide solution in one X mops buffer, seven molar urea at 100 microliters of 10%ammonium per sulfate, and 10 microliters of tetraethyl ethylene diamine and rapidly mix the solution immediately.

Pour the gel between clean glass plates and insert the wellcome carefully avoiding the formation of bubbles. Let the gel polymerize at room temperature for 45 minutes before use. Next Eloqua, 0.5 to 20 micrograms of total RNA For each sample, add an equal volume of acrylamide blue dye to each sample.

Then denature the samples by heating at 80 degrees Celsius for five minutes and cool them on ice until loading. Remove the glass plates from the casting support and assemble them in the electrophoresis cell. Fill both the inner and outer chamber with 500 milliliters of running buffer or rb.

Next, gently remove the comb and pre-run the gel at 200 volts for 10 minutes. Then square RB through a syringe to wash away urea deposits from the wells. Then use flat tips to carefully stratify samples from the bottom of each well.

After the samples, enter the gel at 100 volts for five minutes. Increase the voltage to 200 volts. Cut six pieces of blotting paper to fit the blotting area and an equivalent piece of uncharged nylon membrane.

Dampen the paper sheets and the membrane in RB and completely soak the pads by repeatedly squeezing them in rb. Next, remove the gel from the apparatus and open the glasses with the spatula. Use a clean cutter to remove the wells.

Place a sheet of wet watman paper on the gel and gently lift it up. Place the nylon membrane on the free face of the gel and mark a corner to orient the filter according to sample loading. Complete the sandwich by placing three pieces of paper on each side.

Then roll a plastic rod on the blot surface to remove possible air bubbles. Place the blot between two blotting pads and place the blotting pads in the cassette. Assemble the cassette in the blotting module.

Build the chamber with rb and check that the nylon membrane is between the gel and the positive terminus. Then transfer the RNA at 20 volts for 20 minutes. Synthesize the probe by setting up a phosphate forward reaction on 10 picaMoles of specific antisense oligonucleotide as described in the accompanying text protocol.

Prepare six milliliters of fresh cross-linking solution or XLS. Then saturate a 10 by 10 centimeter piece of blotting paper in the XLS. Next, dismantle the blot and place the membrane on the web.

Three mm. Take care that the RNA is not in direct contact with the saturated paper and place the filter and paper between the two glass plates and wrap them in saran wrap. Then keep the membrane at 60 degrees Celsius for two hours.

Then wash the membrane with doubly distilled water. Preheat 10 milliliters of hybridization solution or HS at 37 degrees Celsius. Denature one milligram of salmon sperm at 95 degrees Celsius for five minutes.

Cool on ice and add to the hs. Then incubate the filter in HS at 37 degrees Celsius for one hour with rotation in a hybridization oven. Replace the exhausted hs with a fresh 10 milliliter aliquot.

Heat the probe at 95 degrees Celsius for 10 minutes. Cool it on ice and add the probe to a novel hs. Incubate the filter at 37 degrees Celsius overnight.

Following overnight incubation. Rinse the filter in washing buffer and wash it thoroughly for 10 minutes at room temperature. Then use a Geiger Mueller detector to check for residual radioactivity at the filter corners.

When the background emission is around five CPS, proceed to membrane exposition. Expose the filter on a molecular imager screen. Use digital conversion to reveal signals.

The enhanced northern blot or ENB ensures higher sensitivity with respect to the standard northern blot or SNB as illustrated by this figure of the auto radiographic detection of the P-I-R-N-A Rasi four. Along a titration of total RNA extracted from adult fly testes. The signal proportionally increases with the amount of fractionated RNA indicating that the dose response ratio lies in a linear range of detection.

The method successfully applies to long RNAs as well as five s ribosomal, RNA, provided that they get a free five prime and a length suitable for acrylamide fractionation. This unanswered block technique makes profiling RNA expression from small samples less than one microgram possible with an average 10 30 fold improvement of RNA, target detection, respect to standard versions as we obtained in our conditions.