Participants: assistant professor Denica Daskalova 

The name of the visited institution - UniStem - Laboratory of Biomedical Embryology, Center for Stem Cell Research, University of Milan, Italy - prof. F. Gandolfi 

Duration: 01.05.2011 - 07.05.2011 

Purpose of the visit – Training and experiments on the topic „RNA and DNA isolation and PCR analysis" 

The research program included:

2 May - еxamination of the laboratory of Biomedical Embryology and other laboratories in campus. I was aware of the available equipment for PCR analysis. I was give a literature on main techniques for pouring gels, design and selection of primers, methods of isolation of RNA and DNA , aseptic handling, storage of samples and etc. PCR test was performed on cDNA from ovarian tissue of sheep. The sample is isolated and stored at -20⁰C. FOR_GAPDH и REV_GAPDH primers were used. Visualization of the PCR product was made on 2% agarose gel.

3 May – isolation of tRNA from sheep ovarian tissue. The procedure lasts 2 days. Were thawed ovarian tissues of sheep stored at -80⁰C. After thawing, the sample was cut very well and added Trizol-reagent. Here are some procedures for centrifugation of sample and adding of chloroform and isopropanol. After good mixing, the sample was left overnight at -20⁰C, for better precipitation.

4 May – second stage of the procedure of RNA. Samples were thawed and centrifuged for 10 min/ 4000 rpm. Supernatant was removed and was added EtOH, centrifuged again and H2O-DEPC was added. The samples were put at 55⁰C for 10 min. 2% agarose electrophoresis of isolated RNA was performed and the quantity was spectrophotometrically measured.Next step was to obtain a cDNA. To isolated samples was added Н₂О, Mix1 (OligoDT,dNTP), Mix2 (Buf.5x, MgCl2,DTT, RnasiOut) и Super Script II ( in termoblok for - 1h/42⁰С – 15 min/ 70⁰С - 4⁰С). The receiving cDNA was stored at -20C.

5 May – PCR analysis of the receiving DNA was made. The following primers of relevant genes: REV_p53, FOR _p53, FOR _HSP-70, REV _HSP-70 were used.

6 May - PCR analysis of the receiving DNA was performed. The following primers of relevant genes: REV_28s, FOR _28s were used.