リアルタイムPCRによる沿岸堆積物および定量16SのrRNA遺伝子と転写物からの同時DNA-RNA抽出

The overall goal of this procedure is to extract high yield and quality DNA and RNA from coastal marine sediments and subsequently quantify 16S ribosomal RNA genes and transcripts. This method describing DNA and RNA extraction from marine sediments followed by quantitative PCR is a key step in the molecular analysis of microbial abundance and diversity in the environment. The main advantage of this technique is that it can be used to coextract DNA and RNA from the same sample which can then be used for downstream analysis, such as quantitative PCR.

Demonstrating the procedure will be Dr.Enrico Tatti from NUI Galway, and Duncan Sweeney, a PhD student from the University of Essex. To coextract DNA and RNA from coastal sediment after preparing RNA free materials and collecting coastal sediment samples according to the text protocol, transfer 0.5 grams of sediment to a two milliliter bead lysing tube. Add 500 microliters of CTAB phosphate buffer and 500 microliters of phenol:chloroform:isoamyl alcohol to the tube and invert five to 10 times to homogenize the sample.

Vortex at full speed for 2.5 minutes before spinning at 16, 000 times g and four degrees Celsius for 10 minutes. Then, in a fume hood, extract the top aqueous layer and transfer it into a new sterile 1.5 milliliter microcentrifuge tube. Next, place the samples on ice and add 500 microliters of ice cold chloroform:isoamyl alcohol.

Then invert the tube several times until an emulsion is visible and centrifuge as before. In a fume hood, extract the top aqueous layer and place in a new sterile 1.5 milliliter tube. Then, to precipitate the nucleic acids, add two volumes of 30%PEG sodium chloride solution and mix well.

Incubate on ice for two hours, or leave the samples at four degrees Celsius overnight. After the incubation, centrifuge the samples at 16, 000 times g at four degrees Celsius for 20 minutes. Using a micropipette, gently remove the supernatant, making sure not to disturb the pellet.

Leave approximately 10 microliters of PEG solution in the tube and add one milliliter of ice cold 70%ethanol. After spinning the sample, use a micropipette to slowly remove the ethanol, taking care not to touch the pellet. Then centrifuge for five seconds and remove the residual ethanol.

Allow the pellet to air dry for five minutes before using 50 microliters of DEPC treated water to resuspend it. Aliquot a 15 microliter sample for DNA and 30 microliters for RNA. The most critical step is to ensure that the DNA/RNA pellet is not dislodged while removing the supernatant and no residual ethanol remains before resuspending the pellet in water, as inhibits downstream molecular reaction.

To digest the DNA in the RNA sample, add three microliters of DNase I buffer and 1.5 microliters of DNase I to the 30 microliter nucleic acid aliquot. Incubate at 37 degrees Celsius for 30 minutes. Mix the DNase inactivation reagent by vortexing for 30 seconds.

Then add 4.8 microliters of the solution to the sample and incubate at room temperature for five minutes, occasionally tapping the bottom of the tubes to mix the solution. After spinning the sample, transfer the supernatant containing RNA into a new tube, taking care not to transfer the precipitant. To check the RNA quality, dilute two microliters of RNA, one in 10, by adding 18 microliters of sterile RNase-free deionized water.

In separate PCR reactions, add one microliter of neat or one in 10 dilution of RNA to a sterile 0.5 milliliter PCR tube. Following the table in the text protocol, add the PCR reaction components to amplify the 16S ribosomal RNA gene. Include a positive control, a PCR inhibitor control, and a negative control.

Then run the PCR reactions using the following amplification parameters. To generate first strand cDNA from RNA, add the reaction reagents to a 0.2 milliliter PCR tube according to the text protocol. Incubate the samples at 65 degrees Celsius for five minutes.

Then place the samples on ice for at least one minute before incubating at 25 degrees Celsius for 10 minutes. After the incubation, add the reagents in the table listed here from the text protocol and incubate at 55 degrees Celsius for 50 minutes. Then inactivate the reaction at 72 degrees Celsius for 10 minutes.

After generating DNA and RNA standards for qPCR according to the text protocol, thaw environmental DNA and cDNA samples, standards, and qPCR reagents on ice. Dilute the nucleic acid standards and prepare one in 10 dilutions of the environmental DNA and cDNA from neat to 10 to the minus three. Make a master mix for the total number of reactions plus 10%and mix well.

Add 19 microliters of master mix and one microliter of template in triplicate to each well in a 96 well optical qPCR plate. Use a qPCR optical cover to cover the plate. Then briefly centrifuge the plate and load it into the heating block of the qPCR machine.

Next, open the qPCR manager software, then click the Protocol tab, click Create New, and add the following amplification conditions. Set the sample volume to 20 microliters, click OK, and save the protocol. Now choose the Plate tab.

Then, under Edit Selected, click Select Fluorophore. Add the reporter dye for the appropriate probe and click OK.Highlight wells containing the standards, then from the Sample Type menu select Standard. Choose Replicate Series to change Replicate Size to three and click Apply.

Choose Dilution Series to add the calculated starting concentration for standard one. Indicate the dilution factor of 10, select Decreasing or Increasing depending on the order the standard curve was added to the plate, and click Apply. Highlight the well positions containing the unknown samples and select Unknown from the Sample Type drop-down menu.

Choose Replicate Series and change Replicate Size to three, then click Apply. Under Sample Name, edit the sample names, and repeat the procedure for no template control wells. Then, on the plate editor box, click OK and save the file.

Then click Start Run. When the run is complete, choose the Quantification tab to display the standard curve, standard curve descriptors, and amplification plots. Finally, choose the Quantification Data tab for Cp values and corresponding starting quantity for genes of unknown samples.

Determine the number of gene copies per gram of wet weight sediment according to the text protocol. As shown in this gel image, a successful nucleic acid extraction yields clear DNA and RNA bands where sharp 23S and 16S ribosomal RNA bands are visible in addition to the presence of a high molecular weight genomic DNA band. This figure demonstrates that when a tenfold dilution range is correctly prepared and amplified to generate a standard curve to be used for the qPCR reaction, a 3.3 cycle difference between each standard dilution is seen.

It is important to report crossing point, or Cp values, from the no template control, or NTC, if present. If this occurs, a Cp cutoff for standard and unknown samples of 3.3 cycles or a log fold higher than the Cp value of the NTC is imposed as previously described. Here, environmental sample amplification from neat, 10 to the minus one, 10 to the minus two, and 10 to the minus three dilutions were carried out.

The neat sample did not amplify and the Cp values of 10 to the minus one to 10 to the minus three dilutions were 24.12, 26.02, and 28.40 respectively. As seen in this graph, standard curve descriptors, including the R squared values of 0.99 and PCR efficiencies within the range of 90 to 110%are desired. In this experiment, the NTC Cp was 30.5, and a negative Cp cutoff of 27.2 was imposed.

Converting gene abundances to per gram of wet weight sediment resulted in 2.5 times 10 to the seventh and 7.1 times 10 to the seventh for the 10 to the minus one and 10 to the minus two dilutions respectively. The 10 to the minus three dilution Cp was greater than the NTC cutoff. In this case, the 10 to the negative two was selected as the optimal template dilution.

While attempting this procedure, it's important to remember that RNA can be rapidly degraded, therefore it's important to keep sample on ice and use gloves to avoid contamination with RNase all the time. Don't forget that we are working with phenol:chloroform and isamyl alcohol, and that can be extremely hazardous. And precautions such as wearing lab coat, gloves, and protective glasses and working under a chemical hood should always be taken while performing this procedure.

After watching this video, you should have a good understanding of how to extract DNA and RNA and quantify gene and transcript abundances in marine coastal sediments. Remember, this procedure can be easily used to extract DNA and RNA and to quantify a range of gene targets from most environmental samples, including soil.