To start the RNA isolation and to attain sufficient yields, 50 zebrafish embryos are pulled into a mortar micro fused tube. The RNA is first extracted using triol, reagent and chloroform after being homogenized thoroughly. The RNA is then cleaned and purified using the Qiagen RNEZ minikit to yield high quality non degraded total RNA for future applications and long-term storage.
The RNA is synthesized into the more stable CD NA product. The cDNA is purified to remove the original RNA template strand. Hello, my name is Sam Peterson from the laboratory of Dr.Jennifer Freeman in the Department of Health Sciences at Purdue University.
Today we'll be showing you how to extract high quality RNA from whole zebrafish embryos and turn it into the CDNA product. Typically, we use this procedure to study gene expression profiles. So let's get started.
When working with RNA, it is very important to work in an environment that is free of RNAs. Use simple precautions such as having reserved pipe pets for use only with RNA procedures and spraying the work area with RNAs away before starting extraction of total RNA To start the extraction work in the fume hood and pull 50 zebrafish embryos in a 1.5 milliliter micro fuge mort tube. Remove as much water as possible and immediately add 250 microliters of triol reagent, which contains phenol.
Use a pellet pestle to lies and homogenize the embryos for approximately 20 strokes until the tissue is sufficiently disrupted. Then add 750 microliters of triazole reagent and incubate for five minutes at room temperature to permit complete dissociation of nuclear protein complexes. When the five minutes are up, add N 0.2 milliliters of chloroform mixed by rocking the tube for 15 seconds and incubate the sample for two minutes at room temperature, then centrifuge at 12, 000 G for 15 minutes at four degrees Celsius.
The mixture will separate into a lower red phenol chloroform phase, an interphase, and a colorless upper aqueous phase. The top layer is approximately naught 0.6 millimeters and it contains the RNA transfer the entire top aqueous layer, approximately naught 0.6 millimeters into a new micro FUZE tube. Being careful not to transfer any of the interphase layer next to precipitate the RNA add 0.5 milliliters isopropanol mix.
And let's sit at room temperature for 10 minutes, then centrifuge the sample at 12, 000 G for 10 minutes at four degrees Celsius. The RNA should form a gel-like pellet. Remove the supernatant without disturbing the pellet and wash by adding one milliliters of 75%ethanol mixed by gentle inversion centrifuge at 7, 500 G for five minutes at four degrees Celsius.
After centrifugation, remove the ethanol and invert the tube to dry for 10 minutes. Resuspend the pellet by adding 100 microliters of RNAs free water and incubating at 55 degrees Celsius for 10 minutes. Finger vortex frequently to aid in RNA rehydration.
Now that the RNA is extracted from the embryos, proceed to RNA cleanup and D a's treatment. To clean the RNA, start with a Rogen RN easy mini kit. Upon first use add four volumes of 100%ethanol to one volume of buffer RPE working in the fume hood.
Add 10 microliters of beta me capto ethanol for every one milliliter of buffer RLT. Then add 350 microliters of buffer RLT and 250 microliters of 100%ethanol to the sample mixed by pipetting up and down several times and transfer the entire sample onto an R and easy column that is loaded into a two milliliter collection tube. Centrifuge at 8, 000 G for one minute at room temperature after centrifuging, discard the flow through and add 700 microliters of buffer RW one to the spin column.
Centrifuge again at 8, 000 G for one minute and discard the flow through to remove DA from the sample. Follow with the DNA's treatment using the RNAs free DNA's kit. Upon receiving the kit, add 550 microliters of nuclease free water to the DNAs and mix gently by inverting to store the stock solution divided into 10 microliter, aliquots and freeze at minus 20 degrees Celsius.
Add 70 microliters of buffer RDD to 10 microliters of DNA's one stock solution mixed by gentle inversion centrifuge briefly and add directly to the R and easy spin column A.Incubate the DNA's treatment for 30 minutes at room temperature. Then add 350 microliters of buffer RW one to the spin column centrifuge at 8, 000 G for one minute at room temperature, discard the flow through and add 500 microliters of buffer RPE to the spin column. Incubate the sample for five minutes at room temperature.
Centrifuge again at 8, 000 G for one minute at room temperature, discard the flow through and add 500 microliters of 75%ethanol to the spin column. Repeat centrifugation at 8, 000 G, but for two minutes at room temperature after centrifugation, discard the flow through and allow the column to dry for five minutes immediately. Centrifuge at 8, 000 G for five minutes at room temperature to remove any leftover tracers of ethanol.
Now transfer the spin column into a new 1.5 milliliter collection tube and add 10 microliters of nuclease free water incubate for one minute to elute the RNA centrifuge at 10, 000 G for one minute. At room temperature, the RNA will be in the flow through. Repeat the elution with another 10 microliters of water.
Check the quantity and quality of the RNA using a NanoDrop ND 1000 spectrophotometer following the manufacturer's instructions actions. In addition, run a denaturing gel or use an Agilent 2100 bioanalyzer to check the integrity of the RNA. If the RNA quality and quantity are satisfactory, move on to CDNA synthesis.
Finally, store the RNA at minus 80 degrees Celsius until CD NA synthesis can be performed. Start the CD NA synthesis by mixing and briefly centrifusion each component of the RNA primer. Mix the RNA DN NTPs and oligo dt.
Add each component to a sterile naut 0.5 milliliter micro fused tube to a total of 10 microliters. For specific volumes, see the accompanying written protocol. Then incubate the sample at 65 degrees Celsius for five minutes.
Immediately transfer the sample to an ice slurry for 10 minutes while the sample is calling, prepare a buffer master mix to a total of nine microliters as outlined in the accompanying written protocol. After cooling, combine the buffer master mix with the RNA primer mixture mixed gently, and collect by brief centrifugation. Next, put the combined sample to incubate in the thermocycler at 42 degrees Celsius for two minutes.
To start the reverse transcription, add one microliter of superscript two RT to the tube and mix. Continue to incubate at 42 degrees Celsius for an hour. Terminate the reaction at 70 degrees Celsius for 15 minutes.
Then chill the sample to four degrees Celsius and transfer to an ice bath on ice. Add one microliter of RNAs H to the sample and incubate for 20 minutes At 37 degrees Celsius, RNAs h degrades the RNA template leaving only the single strand CD NA product cDNA synthesis is now complete, requiring only an additional cleanup and concentration step. To isolate the CD NA product, prepare a 1.5 milliliter phase lock gel tube by centrifugation at 12, 000 G for two minutes.
All of the gels should be at the bottom of the tube. Add 81.5 microliters of tris saturated phenol buffered to pH eight point naught, 81.5 microliters of 24 to one chloroform isol Amal alcohol and the sample into the phase lock gel tube mixed several times by gentle inversion centrifuge the sample at 12, 000 G for five minutes. At room temperature, the CDNA should be in the upper aqueous phase.
Transfer the upper phase, which should be about 20 microliters into a clean 1.5 milliliter micro tube. To precipitate the CDNA, add two microliters of three molar sodium acetate at pH 5.2 and mix gently. Add seven microliters of five milligrams per milliliter of glycogen and mix gently.
Then add 30 microliters of isopropanol and mix gently and allow the sample to incubate at room temperature for 10 minutes. Then centrifuge at 12, 000 G for 20 minutes at room temperature. After centrifugation, a small CDNA pellet appears on the bottom of the tube.
Carefully remove the supernatant with a perpet. Wash the pellet by adding 500 microliters of 70%ethanol and mix by inversion centrifuge the sample at 12, 000 G for five minutes. After centrifugation, remove the SUP natant with ape.
Repeat the wash, add ethanol centrifuge and remove the SUP natant. After the second wash, briefly spin down the tube to gather any remaining liquid. Remove any excess liquid and allowed the pellet to dry at room temperature for five minutes.
Then add 20 microliters of nuclease free water to rehydrate the pellet. Place the sample at 55 degrees Celsius for five minutes. For the rehydration, the CD NA can be stored at minus 20 degrees Celsius.
Finally, check the quantity and quality of the CD NA product using a nano drop ND 1000 spectrophotometer following the manufacturer's instructions, extraction and cleanup of RNA from 50 zebrafish embryos routinely yields approximately 15 micrograms of high quality total RNA as assessed using a NanoDrop ND 1000 spectrophotometer, the two 60 to two 80 absorbance ratio is around 1.9 to two point naught. The integrity of the extracted RNA is evaluated using a denaturing RNA gel. The RNA appears as a smear with two bright bands corresponding to 18 s and 28 s ribosomal RNA.
The 28 s band should be approximately twice as intense as the 18 S band. Alternatively, RNA integrity is evaluated using an Agilent 2100 bioanalyzer. Two sharp peaks corresponding to the 18 s and 28 s ribosomal RNA are visible.
The 28 s peak should be larger than the 18 s peak. Reverse transcription of five micrograms of total RNA routinely yields one to two micrograms of CD NA as assessed using a NanoDrop ND 1000. The two 60 to two 80 absorbance ratio of the CDNA is around 1.8.
We've just shown you how to isolate total RNA from whole zebrafish embryos and convert that RNA into the CD NA product. When doing this procedure, it's important to remember to maintain an environment free of RN ais. So that's it.
Thanks for watching and good luck with your experiments.
