266
is distributed equally across the slide. Take care to not touch
the slide!- Avoid dropping the array slide onto the gasket slide as you may
cause mixing between gasket wells. - Recommended controls for this reaction should include a well
where water replaces RNA in the reverse transcription reaction
and wells with no reverse transcriptase as negative control.
However, you can adjust your controls according to your
experiment design and aims. - Prior to opening the reverse transcription primer tubes, you
should thaw them on ice. Subsequently, mix and centrifuge
them. - In the negative reverse transcriptase batch, replace the reverse
transcriptase volume with RNase-free water. - Reverse transcription master mix should be kept on ice while
you prepare your microRNA samples. - Adjust these volumes for the appropriate number of reverse
transcription reactions and prepare separate batches for experi-
mental miRNA and reference miRNA. - You can use the amplification plots of the reference miRNA
reaction to ensure that the assay is performing correctly.
References
- Ambros V (2004) The functions of animal
microRNAs. Nature 431:350–355. https://
doi.org/10.1038/nature02871 - Mamoori A, Gopalan V, Smith RA, Lam AK
(2016) Modulatory roles of microRNAs in the
regulation of different signalling pathways in
large bowel cancer stem cells. Biol Cell 108:51–
64. https://doi.org/10.1111/boc.201500062 - Salajegheh A, Vosgha H, Rahman MA, Amin
M, Smith RA, Lam AK (2016) Interactive role
of miR-126 on VEGF-A and progression of
papillary and undifferentiated thyroid carci-
noma. Hum Pathol 51:75–85. https://doi.
org/10.1016/j.humpath.2015.12.018 - Gopalan V, Islam F, Pillai S, Tang JC, Tong
DK, Law S, Chan KW, Lam AK (2016)
Overexpression of microRNA-1288 in esopha-
geal squamous cell carcinoma. Exp Cell Res
348:146–154. https://doi.org/10.1016/j.
yexcr.2016.09.010 - Gopalan V, Smith RA, Lam AK (2015)
Downregulation of microRNA-498 in colorec-
tal cancers and its cellular effects. Exp Cell Res
330:423–428. https://doi.org/10.1016/j.
yexcr.2014.08.006
6. Chruscik A, Lam AK (2015) Clinical patho-
logical impacts of microRNAs in papillary thy-
roid carcinoma: a crucial review. Exp Mol
Pathol 99:393–398. https://doi.
org/10.1016/j.yexmp.2015.08.013
7. Salajegheh A, Vosgha H, Md Rahman A, Amin
M, Smith RA, Lam AK (2015) Modulatory role
of miR-205 in angiogenesis and progression of
thyroid cancer. J Mol Endocrinol 55:183–196.
https://doi.org/10.1530/jme-15-0182
8. Ebrahimi F, Gopalan V, Wahab R, Lu CT,
Smith RA, Lam AK (2015) Deregulation of
miR-126 expression in colorectal cancer patho-
genesis and its clinical significance. Exp Cell
Res 339:333–341. https://doi.org/10.1016/
j.yexcr.2015.10.004
9. Gopalan V, Pillai S, Ebrahimi F, Salajegheh A,
Lam TC, Le TK, Langsford N, Ho YH, Smith
RA, Lam AK (2014) Regulation of
microRNA-1288 in colorectal cancer: altered
expression and its clinicopathological signifi-
cance. Mol Carcinog 53(Suppl 1):E36–E44.
https://doi.org/10.1002/mc.21993
10. Ebrahimi F, Gopalan V, Smith RA, Lam AK
(2014) miR-126 in human cancers: clinical
Moein Amin et al.