Quantification of Cellular mRNA Expression

Total RNA was isolated from cell lines and exosomes using the miRCURY RNA Isolation Kit – Cell & Plant (Exiqon, Woburn, MA) followed with spectrophotometry (NanoDrop, Thermo Scientific) for quantification and qualitative analysis. Equal amounts of total RNA was reverse-transcribed by oligo (dT) priming using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA) following the manufacturer's instructions. Real-time PCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) and the SYBR Green PCR Master Mix (Applied Biosystems) as described previously [40] (link). Glut1 and β-Actin primers were purchased from SABiosciences (Frederick, MD).

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Bhattacharya S., Pal K., Sharma A.K., Dutta S.K., Lau J.S., Yan I.K., Wang E., Elkhanany A., Alkharfy K.M., Sanyal A., Patel T.C., Chari S.T., Spaller M.R, & Mukhopadhyay D. (2014). GAIP Interacting Protein C-Terminus Regulates Autophagy and Exosome Biogenesis of Pancreatic Cancer through Metabolic Pathways. PLoS ONE, 9(12), e114409.

Publication 2014

miRCURY RNA Isolation Kit – Cell & Plant NanoDrop iScript cDNA Synthesis kit ABI 7500 Real-Time PCR System SYBR Green PCR Master Mix

Actin Cdna Cell Cell lines Exosomes Glut1 Isolation Oligo Plant Primers Real time pcr Spectrophotometry Sybr green Synthesis

Corresponding Organization : Dartmouth College

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Variable analysis

independent variables

Total RNA isolation method (miRCURY RNA Isolation Kit – Cell & Plant)
RNA quantification method (spectrophotometry using NanoDrop)
Reverse transcription method (iScript cDNA Synthesis kit)
Real-time PCR method (ABI 7500 Real-Time PCR System, SYBR Green PCR Master Mix)

dependent variables

Quantification and qualitative analysis of total RNA
Gene expression levels of Glut1 and β-Actin

control variables

Equal amounts of total RNA used for reverse transcription
Glut1 and β-Actin primers used for real-time PCR

positive controls

None specified

negative controls

None specified

Annotations

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