Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

STUDY DESIGN

16 male Wistar rats were divided into two groups to receive during 5 days daily im injection of:

- Saline 0.6 ml/day, control

- Triamcinolone 80 mg/kg/day

At the end of treatment, diaphragm was removed for further analysis

METHODS

- RNA extraction from diaphragm

- RT-PCR with [alpha-³²P] dCTP using appropriate primers

- Electrophoresis on acrylamide gel

- Radioactivity quantification with PhosphorImager

- Band intensities corrected for CG content

- GAPDH used as house-keeping enzyme

The following SERCA isoforms were measured:

- SERCA 1 expressed in fast-twitch skeletal muscle

- SERCA 2 expressed in slow-twitch skeletal muscle

- SERCA 3 expressed in some non muscular tissues

The isoforms of SERCA 1 and SERCA 2 were also determined:

- SERCA 1a: adult isoform and SERCA 1b: neonatal isoform

- SERCA 2a found in cardiac, slow-twitch and smooth muscles, and SERCA 2b expressed in non muscular tissues

The expression of phospholamban, the regulatory protein of SERCA 2, was also examined

STRATEGY

PCR primers were designed in such a way that they exactly match sequences in the reverse-transcribed cDNA in order to co-amplify SERCA1+SERCA2 and SERCA2+SERCA3. Discrimination between the co-amplified product was subsequently done by using appropriate restriction enzymes (2). Quantification was done on the digested products and data were corrected for the CG content of the amplified sequence.

To measure relative SERCA1a/SERCA1b and SERCA2a/SERCA2b, a different approach was followed based on the fact that the amplified fragments could differ in length due to optional exon.

Quantification of SERCA1a/SERCA1b, SERCA2a/SERCA2b, phospholamban and GAPDH was done on the full-length amplified fragments. The band intensities were subsequently corrected with the data obtained after GAPDH amplification.

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