Authored by Farid E Ahmed*
Introduction
Colorectal cancer (CRC) is the third most common malignancy worldwide, with an estimated one million new cases and half million deaths yearly. Screening for CRC allows early stage diagnosis of malignancy and potentially reduces disease mortality. The convenient and inexpensive fecal occult blood test (FOBT) screening test has low sensitivity and requires dietary restriction, which impedes compliance. Although colonoscopy. Is the golden screening standard for the for this cancer, the invasive nature, abdominal pain and high cost have hampered worldwide application of this procedure. A noninvasive sensitive screen for colon cancer (CC) requiring no dietary restriction is a more convenient test. CC is more abundant in the USA than rectal cancer (RC).The discovery of small non-coding protein sequences, 17- 27 nucleotides long RNAs (such as microRNAs), has opened new opportunities for a non-invasive test for early diagnosis of many cancers. MiRNA functions seem to regulate development and apoptosis, and specific miRNAs are critical in oncogenesis, effective in classifying solid and liquid tumors, and serve as oncogenes or suppressor genes. MiRNA genes are frequently located at fragile sites, as well as minimal regions of loss of heterozygosity, or amplification of common break-point regions, suggesting their involvement in carcinogenesis. Profiles of miRNA expression differ between normal tissues and tumor types, and evidence suggests that miRNA expression profiles can cluster similar tumor types together more accurately than expression profiles of protein-coding mRNA genes. Although exosomal RNA are missed, a parallel carried out on stool miRNAs to compare the extent of loss when colonocytes are only used can be carried out, and an appropriate corrections for exsosomal loss can be made. To ascertain the validity of a miRNA screening test for CC, it must be validated in a study, using a nested case control epidemiology design and employing a prospective specimen collection, retrospective blind evaluation (PRoBE) of control subjects and test colon cancer patients, as delineated by NCI’s Early Detection Research Network (EDRN) http://edrn.nci. nih.gov. Immunoparamagnetic are employed to capture colonocytes from harsh stool environment, whose extracted fragile total small RNA is stabilized shortly after stool excretion by commercial kits so it does not ever fragment, followed by standardized analytical quantitative miRNA dPCR-chip profiling in noninvasive stool samples, to develop a panel of few stable miRNAs for absolute quantitative diagnostic screening of early sporadic colon cancer (stage 0-1), more economically and with higher sensitivity and specificity than other CC screening test on the market today.
A preliminary global microarray expression analysis using an exfoliated colonocytes enrichment strategy, which employed control subjects and various stages (0-4) of CC, using Affymetrix Gene Chip miRNA 2.0 Array, showed 180 preferentially expressed miRNA genes that were either increased (124 miRNAs), or reduced (56 miRNAs) in expression in stool samples from CC patients. This allowed careful selection of 14 miRNAs (12 Up-Regulated, miR-19a, miR-20a, miR-21, miR-31, miR-34a, miR-96, miR-106a, miR-133a, miR-135b, miR-206, miR-224 and miR-302; and 2 Down-Regulated, miR-143 and miR-145) Table 1 for further PCR analysis (Table 1).
Then analysis carried out using absolute miRNAs expression by a chip-based digital PCR by partitioning a sample of DNA or cDNA into many individual, parallel PCR reactions; some of which contain the target molecule (positive), while others do not (negative). A single molecule can be amplified a million-fold or more. During amplification, TaqMan chemistry with dye-labeled probes is used to detect sequence-specific targets. When no target sequence is present, no signal accumulates. Following dPCR analysis, the fraction of negative reactions is used to generate an absolute count of the number of target molecules in the sample, without the need for standards or endogenous controls. In conventional qPCR, the signal from wild-type sequences dominates and obscures the signal from rare sequences. By minimizing the effect of competition between targets, dPCR overcomes the difficulties inherent to amplifying rare sequences and allows for sensitive & precise absolute quantification of the selected miRNAs. Applied Biosystem Quant Studio™ 3D instrument only performs the imaging and primary analysis of the digital chips. The chips themselves must be cycled offline on a Dual Flat Block Gene Amp® 9700 PCR System or the ProFlex™ 2x Flat PCR System. The Quant Studio™ 3D Digital PCR System (Figure 1) can read the digital chip in less than 1 minute, following thermal cycling (Figure 1).
1. Chip Case Lid- The lid used to seal the Digital PCR 20K Chip for thermal cycling and imaging on the Quant StudioTM 3D Instrument.
2. Digital PCR 20K Chip- The 10-mm2 consumable that contains the 20,000 reaction wells, which suspend the individual PCR reactions for thermal cycling and imaging.
3. Quant StudioTM 3D Digital PCR Chip Case- The thermal -conductive base that secures and protects the Digital PCR 20K Chip during all phases of use.
4. Chip ID- A label applies to the Quant StudioTM 3D Digital PCR chip Case Lid that can be used to uniquely identify the chip to which it is applied.
5. Fill Port- The aperture within the Chip Case Lid through which immersion Fluid is injected on to the Chip.
6. Reaction Wells- The 20,000 physical holes within the Digital PCR 20K Chip that suspend the individual PCR reaction.
The current Quant Studio™ 3D Digital PCR Chip allows for one sample per chip; although, duplexing allows for analysis of two targets per chip. Sample prep for digital PCR is no different than for real-time PCR, when using the Quant Studio™ 3D Digital PCR System. The concentration of cDNA stock can be estimated by including all of the necessary dilution factors into the Analysis Suite™ software, which gives the copies/μL in the stock. A critical step in dPCR, is sample partitioning [i.e., division of each sample into thousands of discrete subunits prior to amplification by PCR, each ideally containing either zero or one (or at most, a few) template molecules]. Each partition behaves as an individual PCR reaction –as with real-time PCR—fluorescent FAM probes [or others, as VIC fluorescence. Samples containing amplified products are considered positive (1, fluorescence), and those without product –with little or no fluorescence are negative (0, fluorescence). The ratio of positives to negatives in each sample is the basis of amplification. Unlike real-time qPCR, dPCR does not rely on the number of amplification cycles to determine the initial amount of template nucleic acid in each sample, but it relies on Poisson Statistics to determine the absolute template quantity. The unique sample partitioning step of dPCR, coupled with Poisson Statistics, allows for higher precision than both traditional and qPCR methods; permitting for analysis of rare miRNA targets. The use of a nanofluidic chip provides a convenient mechanism to run thousands of PCR reactions in parallel. Each well is loaded with a mixture of sample, master mix, and Applied Biosystems TaqMan Assay reagents are individually analyzed to detect the presence (positive) or absence (negative) of an endpoint signal. To account for wells that may have received more than one molecule of the target sequence, a correction factor is applied using the Poisson model. It features a filter set that is optimized for the FAM™, VIC®, and ROX™ dyes, available from Life Technologies.
Absolute quantification of the 14 miRNAs is shown in Table 2, and Table 3 is a representation of SDs and R2 for the 14 miRNAs tested by absolute digital PCR. (Figure 2) is Workflow of a digital miRNA’s PCR for colon cancer profiling in human colon tissue or stool samples. (Figure 3). is a graphical representation of the absolute quantification of the 12 up- or 2 down-regulated miRNAs in Human Stool by the QuantStudioTM 3D Digital PCR Chip System. Digital PCR, however, needs a rough estimate of the concentration of miRNAs of interest to carry out first , in order to make appropriate dilutions; Non-template controls and a RT negative control must be set up for each miRNA, when using a “primer pool method” for retro-transcription; a chip-based dPCR method requires less pipetting steps, which reduces potential PCR contamination, and Quant StudioTM 3D chip has 20,000 fixed reaction wells, which reduces variability of dPCR results (Figure 2 & 3 and Table 2 & 3).
To avoid bias and ensure that biomarker selection and outcome assessment will not influence each other, a prospective specimen collection retrospective blinded evaluation (PRoBE) design randomized selection could be employed. An enrichment and exfoliation strategy of colonocytes from stool for miRNA profiling using Dynal superparamagnetic polystyrene beads coated with a mouse IgG1 monoclonal antibody (Ab) Ber-Ep4, specific for an epitope on the protein moiety of the glycopolypeptide membrane antigen Ep-CAM, which is expressed on the surface of colonocytes and colon carcinoma cells, can be used. Comparing the Agilent electrophoretic (18S and 28S) patterns to those obtained from total RNA extracted from stool, and differential lysis of colonocytes by RT lysis buffer (Quagen), could be construed as a validation that the electrophoretic pattern observed in stool (18S and 28S) is truly due to the presence of human colonocytes, and not due to stool contamination with Escherichia coli (16S and 23S). While some exsosomal RNA can be released from purified colonocytes into stool, correction for that effect can be made. Hence, for CRC screening, miRNA markers are more comprehensive and preferable to a DNA-, epigenetic-, mRNA- or a protein-based marker. An added advantage of the use of the stable, nondegradable miRNAs by PCR expression, or chip-based methods is being automatable, making them more economical and acceptable by laboratory personnel performing these assays.
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