Analyzing Unstable MicroRNA Expression

Samples were diluted to 5ng/μl and transcribed into cDNA using a Taqman Advanced MiRNA cDNA Synthesis Kit from Applied BioSciences. The cDNA underwent an additional amplification step to increase yields of unstable miRNAs (MiR-Amp). Samples were diluted 1:10 and loaded onto qPCR plates with Taqman Fast Advanced Master Mix. Each sample received 2 qPCR runs using the StepOnePlus Real Time polymerase chain reaction (PCR) System (Applied Biosystems, Foster City, CA), including one to evaluate for U6 a small non-coding spliceosome RNA that is a common endogenous control (Campos-Melo et al., 2013 (link)). The next qPCR run was with the custom miRNA plates with primers for selected targets. Samples were tested in duplicate.

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Alvia M., Aytan N., Spencer K.R., Foster Z.W., Rauf N.A., Guilderson L., Robey I., Averill J.G., Walker S.E., Alvarez V.E., Huber B.R., Mathais R., Cormier K.A., Nicks R., Pothast M., Labadorf A., Agus F., Alosco M.L., Mez J., Kowall N.W., McKee A.C., Brady C.B, & Stein T.D. (2022). MicroRNA Alterations in Chronic Traumatic Encephalopathy and Amyotrophic Lateral Sclerosis. Frontiers in Neuroscience, 16, 855096.

Publication 2022

StepOnePlus Real Time polymerase chain reaction (PCR) System

Cdna Mirna Primers Real time polymerase chain reaction Small non coding rna Spliceosome Synthesis

Corresponding Organization : VA Boston Healthcare System

Other organizations : Southern Arizona VA Health Care System

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Variable analysis

independent variables

Dilution of samples to 5ng/μl
Transcription of samples into cDNA using Taqman Advanced MiRNA cDNA Synthesis Kit
Additional amplification step (MiR-Amp) to increase yields of unstable miRNAs
Dilution of samples 1:10 before loading onto qPCR plates

dependent variables

Expression levels of selected miRNA targets

control variables

Use of U6 small non-coding spliceosome RNA as an endogenous control
Duplicate testing of samples

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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