Please direct all comments, questions, and suggestions to biobzc@hofstra.edu

RT-PCR DEMONSTRATION OF ALTERNATIVE SPLICING

The myosin heavy chain gene provides an excellent example of the alternative splicing of exons to produce different gene products at different times during development and in different tissues. For example, the gene contains several alternative splicing patterns for  exons 3, 7, 9, 11and 15. Exons 3a, 7a, 9c, 11d and 15 b are expressed in embryonic somatic muscle, whereas exons 3b, 7d, 9a, 11b, and 15a are expressed in adult jump muscle. Several alternate Mhc transcripts are generated in Drosophila by the selective use of alternative polyadenylation sites and by the alternative splicing of exons. Exon 18 is not included in the embryonic and some isoforms of the adult transcript, but is included in other isoforms of the adult message. We will demonstrate this alternative splicing using RT-PCR (reverse transcription /polymerase chain reaction). First we will convert mRNA from wild type adult flies and wild type embryos into cDNAs using oligo dT as the primer for reverse transcription. We will then amplify part of the resulting cDNAs using PCR primers from exons 17 and 18. A product is obtained from the adult cDNA sample but no product is obtained from the embryonic cDNA sample.

Picking the Primers

You will first use sequence available in Flybase or the NCBI database to pick primers for the PCR reaction. Use cDNA or mRNA sequence as template when picking your primers. Why wouldn't you want to use genomic sequence to pick the primers? What special manipulation do you need to do to the sequence if you use mRNA as a template to pick your primers? You may use any of the software we have used in class to pick your primers. Pick a forward primer from Exon 17 and a reverse primer from exon 18 of the Mhc gene¹. As an internal control for cDNA quality, b actin standard primers (Ambion) will also be used to amplify a product from each cDNA pool.

RT

We will be using the Enhanced Avian RT-PCR Kit from Sigma for the RT-PCR; we will use a two-step process since we will use different primers for the two parts of the procedure. We will use the oligo (dT) primer for the reverse transcription

RT Reaction

1. Add the following reagents to a thin-walled 500µl microcentrifuge tube on ice:

2. Mix gently and briefly centrifuge to collect all components to the bottom of the tube.

3. Place the tube in the water bath at 70-85˚ C for 10 minutes.

4. Place the tube on ice and add the following components:

5. Incubate the reaction at 55˚C for 50 minutes. Typically this reaction would be run at 42-50 ˚C if we were using a different template. We are using RNA as a template. RNA has a considerable amount of secondary structure, therefore, a higher temperature is used in the transcription step. 

PCR

We will use the primers that you picked for the PCR reaction. Set up the following 50 µl PCR reaction:

Add 100µl sterile nucleotide free mineral oil. Place in the thermal cycler. Set the cycler for the following sequence of steps

94˚C - 5 min

35 cycles of

Run 10µl of the PCR product on an agarose gel. Store the PCR product at -20˚C.

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¹Primers that have been used successfully: