Quantifying SMN Isoforms in Duodenum

Duodenum samples were homogenized using a Precellys Homogenizer (Berlin Technologies) in RLT lysis buffer using RNeasy Mini Kit (Qiagen, Chatsworth, CA). Total RNA extraction and reverse-transcription were performed as described previously [10 (link),31 (link)]. The ratio of full-length SMN2 to Δ7 SMN2 transcripts was quantified by quantitative real-time RT-PCR. The expression of human SMN protein was measured by western blotting as previously described [31 (link)]. β-tubulin was used as loading control in all samples. Blots were developed with an enhanced chemiluminescence detection kit (Bio-Rad, California, USA). Semi-quantification of band intensity was analyzed by imageJ software.

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Sintusek P., Catapano F., Angkathunkayul N., Marrosu E., Parson S.H., Morgan J.E., Muntoni F, & Zhou H. (2016). Histopathological Defects in Intestine in Severe Spinal Muscular Atrophy Mice Are Improved by Systemic Antisense Oligonucleotide Treatment. PLoS ONE, 11(5), e0155032.

Publication 2016

RNeasy Mini Kit enhanced chemiluminescence detection kit

Buffer Chemiluminescence Duodenum Protein human Quantitative real time pcr Reverse transcription Tubulin

Corresponding Organization : University College London

Other organizations : King Chulalongkorn Memorial Hospital, Chulalongkorn University, Ramathibodi Hospital, Mahidol University, University of Edinburgh, University of Aberdeen

Top 5 similar protocols

Variable analysis

independent variables

Homogenization method (Precellys Homogenizer)

dependent variables

Ratio of full-length SMN2 to Δ7 SMN2 transcripts (quantified by qRT-PCR)
Expression of human SMN protein (measured by western blotting)

control variables

RLT lysis buffer used for homogenization
RNeasy Mini Kit used for total RNA extraction
β-tubulin used as loading control for western blotting

controls

Positive control: Not specified
Negative control: Not specified

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