Question

Preparation of paraffin section

Answer 1

Section paraffin blocks at the desired thickness (usually 4-5 µm) on a microtome and float on a 40°C water bath containing distilled water.

Transfer the sections onto a Superfrost Plus slide. Allow the slides to dry overnight and store slides at room temperature until ready for use.

Answer 2

Formaldehyde fixation followed by dehydration and paraffin embedding (FFPE) is commonly used to preserve tissue specimens for histological studies and provides most archival tissue samples. The isolation of high-quality RNA from FFPE tissues remains a challenge for molecular studies, despite the availability of multiple published and commercial methods.1 RNA degradation and formaldehyde modification of RNA appear to be the major contributors to this challenge. Degradation of RNA to low molecular weight species may be because of either sample treatment before and during fixation2 or long-term (1 year or longer) storage in paraffin.3 RNA extracted from FFPE tissues is usually fragmented to an average of 100 bases in length.4,5 Reproducible RT-PCR on FFPE-extracted RNA is limited to amplicons of fewer than 300 bases.6 Most laboratories strive to amplify segments of 150 or fewer bases. Degraded RNA can often be quantified by techniques that use short oligonucleotides, such as microarray and micro-RNA analyses, and by RT–quantitative PCR (qRT-PCR), but the results are almost invariably less sensitive and less reproducible than achieved using RNA extracted from fresh or fresh-frozen sources.7,8