This method permits to detect, by a non-invasive approach, patient with colorectal neoplastic lesion. In particular, this test permits, in association with tests currently used in colorectal cancer screening program, to better predict the presence of cancer or higher risk of abnormal lesions. To begin this protocol, centrifuge an aliquot of each positive control, standard DNAs, and spike-in DNA, in a dry format for 10 seconds on a mini centrifuge.
Resuspend the positive control with 750 microliters of water. Resuspend the spike-in DNA, used as an exogenous internal control, to test the presence of inhibitors, with 100 microliters of water. For the standard curve, resuspend the dry standard with 100 microliters of water and make four 1:5 dilutions starting from the stock solution.
Then, carefully vortex the reagents for 15 seconds, and centrifuge in a mini centrifuge for 10 seconds. For a complete resuspension, store the liquid reagents at room temperature for 30 minutes before using. To prepare the 1x spike-in DNA control, mix five microliters of FL-DNA spike with 45 microliters of sterile water.
For the samples, mix 75 microliters of each sample with 50 microliters of 1x spike-in DNA, yielding a total volume of 125 microliters. First, open the qPCR operating software. To set up the plate, set the Well Type for all eight positions in column one, as Standard.
Set the Well Type for the A2 and B2 wells as NTC. Set the Well Type for the positive controls, C2 and D2, as Unknown. And set the Well Type for all other positions as Unknown.
Select all 96 positions. Add the dyes, FAM and HEX and click Sync Plate. Then set the Thermal Profile, as provided.
Prepare the desired number of 8-well strips containing complete lyophilized amplification mixtures, with specific primers and probes targeting the human DNA, and the internal control. To bring the contents to the bottom of the tubes, centrifuge the strips for 10 seconds. Gently remove the seals from the strips, making sure not to lose any pellet.
Then add 25 microliters of water to the negative control wells. 25 microliters of sample DNA to sample wells and 25 microliters of positive control DNA to the positive control wells. For the standard curve, add 25 microliters of standard 1, 2, 3, and 4 in each dedicated well.
Use 8-strip flat optical caps to carefully close all the strips and vortex for a few seconds. Centrifuge the strips for 10 seconds, load them into the instrument, and start the run. At the end of the run, for FAM FL-DNA A and HEX IC, select columns, A, C, E, and G.For FAM FL-DNA B and HEX IC, select columns, B, D, F, and H.For the standard quantities starting amount, set 10 nanograms per reaction for A1 and B1 wells, two nanograms per reaction for C1 and D1, point four nanograms per reaction for E1 and F1, and point zero eight nanograms per reaction for G1 and H1.Then, for both FAM and HEX channels set Threshold Fluorescence values to 150.
In the box Result Table, click Column Options, then, Select All, and then Okay, to obtain the results in both channels with their respective Cq and delta R last values, which are supplied by the real time qPCR instrument software. Delta R last corresponds to the fluorescence value normalized to the last amplification cycle. In the box Result Table, right click on the table to open the context menu.
Click Send to Excel to export the raw data. And then upload the Excel file on the dedicated analysis software for automated calculation of FL-DNA quantity. From the combination of iFOBT and FL-DNA values estimate for each patient, the colorectal cancer risk.
Fluorescence curves of a suitable positive sample show the sample signal on the HEX channel, which is the internal control, within the acceptable range. Positive signal is above the threshold on the FAM channel, which shows the amplification of target genes. Fluorescence curves of a suitable negative sample show the sample signal within the acceptable range on the HEX channel.
The negative control signal is below the threshold on the FAM channel. On the other hand, curves of a non-suitable sample show the sample signal that is not within the acceptable range on the HEX channel. Most likely due to a potential inhibition.
Therefore, this sample must be repeated, starting from the extraction. Software analysis output table shows data for both Mix A and Mix B in terms of FL-DNA concentration for reaction controls, clinical samples, and run quality control results. Sample number four does not have any results because it didn't pass the quality control.
It must be repeated and reanalyzed. Please be careful when resuspended the DNA for spike-in in control to the standard curve. And when distributing different DNAs to 8-well strips, as well as during molecular detection in the results analysis section.
This method must be performed in combination with the iFOBT test to permit a better evaluation of colorectal neoplastic lesions, it must be carried out with the same fecal sample.
