Quantification of MicroRNAs for the Diagnostic
Screening of Colon Cancer in Human Stool by
Absolute Digital(d)PCR*
Volume 1 - Issue 5
Farid E. Ahmed1*, Mostafa M Gouda2,3, and Nancy C. Ahmed1
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- 1Institute for Research in Biotechnology, Greenville, USA
- 1Department of Nutrition, El-Bohooth Street, Dokki, Cairo, Egypt
- 1National Research and Development Center for Egg Processing, Wuhan, Hubei, PR, China
*Corresponding author:
Farid E Ahmed, Institute for Research in Biotechnology, 2905 South Memorial Drive, Greenville, NC 27834,
USA
Received: January 08, 2019 Published: January 18, 2019
DOI: 10.32474/SCSOAJ.2019.01.000125
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Abstract
There is currently no validated micro(mi)RNA diagnostic stool test to screen for colon cancer (CC) on the market because of the
complexity of fecal density, vulnerability of stool to daily changes, and the presence of three sources of miRNAs in stool (cell-free
from fecal homogenates, exsosomal miRNAs from fecal exosomes, and fecal colonocytes). By employing earlier on a microarray
miRNA experiment, using Affymetrix GeneChip miRNA 2.0 Arrays, on immunocaptured and enriched stool colonocytes of 15
subjects [three healthy controls and twelve colon cancer patients [three TNM stage 0-1 (e.g., polyps 1 cm, villous or tubvillous,
or with high grade dysplasia), three stage 2, three stage 3, and three stage 4] in triplicates, this allowed for selection of a smaller
panel of 14 preferentially expressed mature miRNAs associated with colon cancer (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). Then carrying out an absolute quantitative digital PCR on these 15 stool samples from TNM stages 0-4 on total small
RNA extracted by immunocapture, followed by RT that employed a Custom TaqMan® miRNA Reverse Transcription (RT) Kit and
TaqMan RT Primer Pool, and absolute quantification of miRNAs, in copies/μl, measured using a chip-based Absolute QuantStudio
3D Digital PCR analysis, allowed for validating the microarray results. To ensure that human and not bacterial small total RNA was
chosen, coextraction protocols with E. coli K1 strain RS18 was carried out, followed by comparing Agilent electrophoretic patterns
with human and bacterial electrophoretic patterns, and also random samples were sequenced using mRNA/miRNA sequencing, to
ensure that human and not bacterial mRNA was chosen.
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