This method can help answer key questions in the chronic lymphocytic leukemia field, such as the accurate prognosis of patients. The main advantage of this technique is that it is simple, fast, and highly accurate. The implications of this technique extend towards robust prognostication, most particularly risk certifications with patients with chronic lymphocytic leukemia because the accurate prognostication may later define therapeutic management of the disease.
Visual demonstration of this method is critical, such as the PCR product selection and digest sequence analysis types are difficult to learn because they require both precision and experience. To begin this procedure, add 200 microliters of EDTA to a 50 milliliter sterile collection tube. Add 20 milliliters of peripheral blood and 15 milliliters of RPMI medium to the tube and pipette up and down two to three times to mix.
Next, add 15 milliliters of density gradient medium to a new 50 milliliter collection tube. Slowly layer the peripheral blood solution onto the density gradient medium, making sure that it does not disrupt the gradient. Centrifuge at 800 times G for 20 minutes with the centrifuge brake off.
Then use a sterile Pasteur pipette to collect the mononuclear cell ring that has formed between the upper plasma platelet layer and the density gradient medium layer and transfer it to a new sterile 15 milliliter collection tube. Add RPMI medium such that the final volume is 12 milliliters and pipette the solution up and down to mix. Centrifuge at 800 times G for eight minutes.
Discard the supernatant and re-suspend the cell pellet with 10 milliliters of RPMI medium. Centrifuge again at 750 times G for eight minutes. After this, discard the supernatant and dissolve the cell pellet in one milliliter of PBS.
Using a new Bauer plate, form a cell count by mixing 20 microliters of sample and 80 microliters of one to 10 Turk solution. If downstream processing of the sample is not scheduled for the same day, centrifuge the sample at 750 times G for eight minutes. Discard the supernatant and store the cell pellet at minus 80 degrees Celsius.
First, prepare the primer mix using equimolar quantities of each subgroup primer to ensure unbiased amplification. Mix the PCR reagents as listed in table two of the text protocol. Add 0.5 microliters of TAP polymerase to the PCR tube containing the mixed PCR reagents.
Then, add either 100 nanograms of gDNA or two microliters of cDNA. Run the thermal PCR protocol as detailed in table three of the text protocol. After this, check for PCR products of approximately 500 base pairs when using IGHV liter primers or 350 base pairs when using the IGHPFR1 primer mixes.
To begin PCR product purification, load the total volume of the PCR product onto a three percent low-melting agarose gel. Allow the PCR products to run on the gel until they separate from the background. Excise the sharp prominent PCR bands and then purify and elute them.
If two rearrangements are detected, both bands should be excised and sequenced separately. To perform exo-sap cleanup, follow the manufacturer's instructions regarding the specific volumes to use. Gently vortex each sample to mix and incubate them on a PCR block at 37 degrees Celsius for 30 minutes.
Inactivate the enzymes by heating them to 85 degrees Celsius for 15 minutes. After this, load a small quantity of the PCR products on a three percent agarose gel to assess the purity of the product. The last checkpoint prior to sequencing is determining the clonality of the sample using fragment analysis.
To begin analyzing the sequence, start up the appropriate software. Specify the species, such as homo sapiens, and the receptor type or locus. Pace the sequences to be analyzed in fasta format and including identifiers into the text area in batches of up to 50 sequences per run.
Then, choose one of the following three display options for the results. Detailed view:choose this option in order to get the results for each sequence individually. Synthesis view:choose this option to compile a summary table ordered by IGHV gene and allele name or by sequence input order.
This option also provides an alignment of sequences that in any submitted batch are assigned to the same IGHV gene and allele, and lastly, Excel file. Choose this option to provide all output files as spreadsheets incorporated into a single file. Only the use of IGHV liter primers enables the amplification of the full length of the rearranged IGHV, thus permitting the true HSM load to be determined.
The expected PCR product is approximately 500 base pairs. In rare instances where IGHV liter primers cannot amplify the clonotypic IG rearrangement, IGHBFR1 primers may be used. The expected PCR product is approximately 350 base pairs.
The sequences obtained are then analyzed using the IMG TV Quest tool. The first row in the results table states the functionality of the sequence, as an IG rearranged sequence can be either productive or unproductive. An IGH gene rearrangement is unproductive is stop codons are present within either the VDJ region and/or if the rearrangement is out of frame due to insertions/deletions.
It is important to note that only productive and thus functional rearrangements should be analyzed further. When using the default IMG TV Quest parameters, insertions and deletions are not automatically detected, however strong indications that a sequence may carry such alterations are provided by the tool and a warning alert appears below the result summary table. When attempting this procedure, it is important to remember to land the PCR products on the gel long enough so that the clonal bond can be discriminated from the polyclonal background.
After its development, this technique paved the way for researchers in the field of chronic lymphocytic leukemia immunogenetics to explore the prognostic impact of the somatic epimutation status in patients. Don't forget that working with ethylium bromide can be extremely hazardous and precautions such as working under the hood at all times should always be taken while performing this procedure.
