The key question we want to address with this method is how can we use RNA samples from material purchased at local food markets for the cloning of biotechnologically relevant genes? Here, we use Pacific oysters as a case study for demonstrating this approach and potential further application. The main advantage of this technique is that conventional genome or cDNA sequencing of the oyster samples can be circumvented.
Instead, sequences of the cytochrome c oxidase subunit I and NADH dehydrogenase were compared with publicly available reference sequences from the Pacific oyster to select the most closely related specimen. The cDNA prepared from this specimen can be directly used for the cloning of biotechnologically relevant genes, which can be selected using the public sequence information. Demonstrating the procedure will be Yongmei Lyu and Yuquan Li, two graduate students from the Glycomics and Glycan Bioengineering Research Center at Nanjing Agricultural University.
To prepare oyster tissue sample, use a sterilized scalpel to cut out approximately 100 milligrams of homogeneous soft tissue from the approximate geometric center of each oyster specimen. Transfer the sample into a mortar filled with 50 milliliters of liquid nitrogen. Grind the flash-frozen oyster tissue into a fine powder.
Weigh out 75 milligrams of each specimen's frozen tissue into a sterile 1.5-milliliter centrifuge tube, and mix with one milliliter of guanidinium thiocyanate-phenol extraction reagent. The most critical step of this procedure is the RNA extraction step. In order to minimize RNA degradation, it is essential to reduce the time between harvesting the oyster tissue and the RNA extraction.
Centrifuge the sample at 14, 000 times g and four degrees Celsius for 15 minutes. Transfer the supernatant to a new 1.5-milliliter centrifuge tube, add 200 microliters of chloroform, and mix thoroughly on a vortex mixer for 10 to 15 seconds until the mixture turns milky white. Next, centrifuge for 15 minutes as done previously.
With a 200-microliter pipette, carefully transfer the upper aqueous layer, without disturbing the interphase, into a new 1.5-milliliter centrifuge tube. Add 500 microliters of isopropyl alcohol into the centrifuge tube, and invert gently to mix the samples. Then leave the samples on ice for 20 minutes.
Centrifuge at 14, 000 times g and four degrees Celsius for eight minutes, and remove the supernatant. Resuspend the pellet in one milliliter of 75%ethanol, and centrifuge at 14, 000 times g and four degrees Celsius for five minutes. Remove all of the supernatant, and repeat the wash in ethanol.
Dry the pellets for six minutes at room temperature. Then, dissolve the dried RNA pellet in 25 microliters of DEPC-treated water, and keep the tube on ice. Use the RNA samples within 24 hours.
To generate a cDNA library, first prepare a reaction mixture for each RNA sample in a 300-microliter PCR tube by adding solutions according to the manuscript. Add one microliter of the extracted RNA sample into the tube. Incubate the mixture in a PCR thermocycler for 60 minutes at 42 degrees Celsius, and then increase the temperature to 95 degrees Celsius for five minutes.
Store the generated cDNA library for up to 12 months at minus 20 degrees Celsius. First, add one microliter of the generated cDNA library to tubes containing PCR mixtures, prepared according to the manuscript. Place the PCR tubes into the PCR thermocycler, and perform the PCR amplification with an initial denaturation step and 35 PCR reaction cycles consisting of an annealing step, an elongation step, and a denaturation step according to the manuscript.
After the cycles, perform one finalizing elongation step for five minutes. Use five microliters of the PCR product to verify the quality by agarose gel electrophoresis. Observe the amplified COX1 or ND gene as a single band at either 759 or 748 base pairs, respectively.
To purify the rest of the PCR product, add 100 microliters of DNA-binding buffer containing high concentrations of chaotropic salts to each sample. Vortex briefly to mix the contents. Place a purification column into a two-milliliter centrifuge tube.
Pipette the reaction mixture into the column. Place the tube with the mounted column in a centrifuge at 14, 000 times g for one minute at room temperature. After discarding the filtrate from the two-milliliter centrifuge tube, wash two times with 700 and 400 microliters of washing solution W2 by centrifuging at 14, 000 times g.
Next, heat one milliliter of deionized water to 65 degrees Celsius in a metal block heater. Transfer the column into a new 1.5-milliliter centrifuge tube, and pipette 25 microliters of the preheated deionized water to the center of the white column membrane. Let the membrane soak for one minute at room temperature.
Then, centrifuge the tube with the column at 14, 000 times g for one minute at room temperature. Discard the column, and store the purified PCR product for up to 12 months at minus 20 degrees Celsius. Optionally, the COX1 or ND reverse primers can also be used for bidirectional sequencing.
For Sanger sequencing, use the relevant COX1 or ND forward primer. After retrieving the sequencing results, compare the sequences with the genome sequence of the Pacific oyster reference strain using the NCBI Nucleotide BLAST online tool. Use respective forward and reverse primers of MgUGD and MgUXS genes to amplify and purify by PCR as done previously.
After incubating purified PCR products with digestion buffer, prepare the predigested pET-30a vector by dissolving 500 nanograms of pET-30a in a 1.5-milliliter centrifuge tube, and add deionized water to top up to 16 microliters. Then, add two microliters of 10-times-concentrated digestion buffer and one microliter each of the 20 units restriction endonucleases Nde1 and Xho1 into the tube. Incubate the mixture at 37 degrees Celsius for three hours.
After that, add one microliter of one-unit alkaline phosphatase, and incubate at 37 degrees Celsius for an additional hour. Then, place the tube in a preheated metal block heater at 75 degrees Celsius for 10 minutes to inactivate the alkaline phosphatase. After cultivating E.coli BL21 cells bearing the MgUGD and MgUXS genes, transfer the culture into 400-milliliter LB medium in a two-liter shaking flask, and place it on a shaker at 200 rpm at a temperature of 37 degrees Celsius.
After three hours, check the optical density on a photometer at a wavelength of 600 nanometers, and ensure it reaches an absorption of approximately 0.5. Then reduce the shaker temperature to 20 degrees Celsius. Add 400 microliters of one-molar IPTG to induce the expression of the recombinant proteins for three hours.
After harvesting the cells, disrupt the cells by sonication for 20 minutes at four degrees Celsius. Centrifuge at 14, 000 times g and four degrees Celsius for 20 minutes, and collect the supernatant in a new tube for the activity test. After the activity assay, quench the reactions by adding 20 microliters of methanol and 40 microliters of chloroform to the MgUXS and MgUGD mixtures.
Vortex the sample mixtures, and centrifuge at 14, 000 times g for six minutes and four degrees Celsius. Collect the upper aqueous layer of each tube in new tubes for MALDI-TOF mass spectrometry. In this experiment, sequence alignment of the COX1 and ND gene sequences of a highly divergent specimen were compared with the reference Pacific oyster strain.
The red arrows show nucleotide differences between the reference sequence and the sequence of the obtained cDNA samples. The sequences of the COX1 and ND genes of a closely related oyster specimen show low divergence from the reference Pacific oyster strain. MALDI-TOF mass spectrometry shows successful application of the cDNA library to clone the industrially relevant genes MgUGD and MgUXS.
The gel image also identifies UDP-glucuronic acid and UDP-xylose generated by the enzymatic action of the successively expressed and purified MgUGD and MgUXS. This method does not require a perfect match between the reference sequence and the obtained marker sequences and should give the researcher confidence to work with unreferenced oysters. In principle, this method can be applied to any unreferenced biological sample for which sequences from a closely related reference species are publicly available.
This method permits non-expert researchers to easily access genetic material of potential biological importance and paves the way for more scientists to work with easily accessible but incompletely identified biological samples.
