This protocol aims to obtain high quality cell types specific RNAs from a small number of cells in a complex tissue without cell sorting. It employs a recently available mouse model that allows researchers to isolate a ribosome bound RNAs using GLP pull down. The method is also suitable for isolating ribosome bound RNAs from any EGFP expressing cells.
To begin, add two milliliters of ice cold homogenization working buffer to a glass tissue grinder set. Quickly place the frozen sample into the grinder and homogenize the tissue with 30 strokes on ice using a loose pestle. transfer the homogenate to a two milliliter round bottom tube and centrifuge at 12, 000 g for 10 minutes at four degrees Celsius.
Then transfer the supernatant to a new two milliliter tube without disturbing the pellet, reserving 100 microliters as the input. Incubate the supernatant with the anti-GFP antibody at four degrees Celsius on an end over end rotator overnight. Transfer the homogenate and antibody mixture to a new two milliliter round bottom tube containing washed protein g beads and incubated four degrees Celsius on rotator at 24 RPM for two hours.
Separate the magnetic beads from the supernatant using a magnetic rack. Save the supernatant as the negative fraction which contains the RNAs in EGFP negative cells and the RNAs in EGFP positive cells that are not bound to ribosomes. Add one milliliter of high salt wash buffer to the beads and briefly vortex the tube to wash the beads.
Then place the tube in a magnetic rack. Remove the wash buffer and repeat the washing steps two more times to obtain the beads ribosome RNA complex from EGFP positive cells. Incubate the beads with 50 microliters of extraction buffer from the RNA isolation kit in a ThermoMixer for 30 minutes to release RNAs from the beads.
Separate the beads with a magnetic rack and transfer the supernatant to a 1.5 milliliter tube. Centrifuge the tube at 3000 g for two minutes. Then pipette the supernatant to a new 1.5 milliliter tube.
To pre-condition the RNA purification column, pipette 250 microliters of conditioning buffer onto the purification column and incubate for five minutes at room temperature. Then centrifuge the column at 16, 000 g for one minute. Pipette an equal volume of 70%ethanol into the cell extract and mix well by pipetting.
Pipette up to 200 microliters of the mixture into the preconditioned RNA purification column. To reduce the loss of samples do not fill the column more than 80%of its capacity. Repeat this step several times until all RNAs are collected in the same column of each fraction.
Centrifuge the column for two minutes to bind RNA to the membrane in the column. Then continue centrifugation for 30 seconds. Discard the flow through and pipette 100 microliters of wash buffer one into the column.
Centrifuge for one minute, then discard the flow through. Pipette 75 microliters of DNA solution mix directly into the purification column membrane. Incubate at room temperature for 15 minutes.
Then, pipette 40 microliters of wash buffer one into the column and centrifuge for another 30 seconds. Discard the flow through. Pipette 100 microliters of wash buffer two into the column and centrifuge at 8, 000 g for one minute.
Discard the flow through. Buy PET 100 microliters of wash buffer two into the column and centrifuge at 16, 000 g for two minutes. Discard the flow through, and re-centrifuge the same column at 16, 000 g for one minute to remove all traces of wash buffer.
Transfer the column to a new 1.5 milliliter microcentrifuge tube. pipette 12 microliters of RNase free water directly onto the membrane of the purification column ensuring that the pipette tip does not touch the membrane. Incubate at room temperature for one minute and centrifuge at 1000 g for one minute.
Then at 16, 000 G for one minute to elute the RNA. Use a bioanalyzer to assess the quality and quantity of the extracted RNA. Store the RNAs in a 80 degree freezer or send out for further analysis.
Mice carrying both genetically engineered gene alleles, Gli1-CreERT2 and NuTRAP we're injected with tamoxifen once a day, every other day for three injections. Immunofluorescence analysis of collected tissues show that the EGFP was expressed in interstitial cells in the testes. EGFP was also found in adrenal capsular cells in Gli1-CreERT2, NuTRAP mice.
In Sonic Hedgehog-CreERT2, NuTRAP mice The EGFP positive cell population resides in the outer cortex of the adrenal gland underneath the capsule, confirming the expression of EGFP pre-expressing cells. After the extraction of the cell type specific RNAs, the extracted RNA was sent for microarray analysis. The results showed that about 3000 genes were enriched in the positive fraction, compared to genes in the negative fraction, whereas about 4, 000 genes were enriched in the negative fraction.
Among the differential expressed genes, Leydig cell associated genes Hsd11b1 and Hsd3b6 were enriched in the positive fraction. In the negative fraction, the Sertoli cell associated genes Desert Hedgehog and Gstm6 were enriched. Only a few differentially expressed genes were identified in comparing the negative fraction with the input.
Realtime quantitative RT-PCR was used to confirm the expression of key genes in the positive and negative fractions. Similar to what was found in the microarray assay, steroidogenic enzymes 3-beta hydroxy steroid dehydrogenase and cholesterol side chain cleavage enzyme were enriched in the positive fraction. Meanwhile, the Sertoli cell marker SRY box transcription factor nine and the germ cell marker synaptonemal complex protein three were enriched in the negative fraction.
When attempting this protocol, it is important to prepare fresh homogenization working buffer Add DTT, cycloheximide recombinant ribonuclease, and proteinase inhibitor cocktail to the homogenization stock solution only before use. It is also important to prepare fresh low salt and the high salt wash buffers before use.
