Cytokine profiles in paraffin-embedded biopsy samples of lepromatous leprosy patients: semi-quantitative measure of cytokine mRNA using RT-PCR.
A reproducible technique for fixation of tissue, RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) analysis from paraffin-embedded leprosy biopsies, has been developed and used to study the mRNA profiles. This approach is valuable in retrospective analysis of gene expression, and the handling of infectious biopsy material is also minimized. Among the methods of RNA extraction compared, the most efficient method was found to be incubation of the tissue sections in digestion buffer followed by extraction with Trizol. The experimental conditions were optimized for first strand cDNA synthesis and PCR, and used to measure the quantity of cytokine transcripts in biopsy materials. Interleukin-10 (IL-10) mRNA was detectable in all cases examined, which correlates well with other earlier reports using frozen tissues. However, IL-5 transcripts were present in 60% of the biopsies, unlike the earlier reports which showed IL-5 mRNA in all LL cases. Transforming growth factor-beta (TGF-beta) mRNA was detected in 80% of the biopsies, and this confirmed earlier immuno-cytochemical data which showed TGF-beta protein in all cases. Tumor necrosis factor-alpha mRNA was found in about 60% of the biopsies; whereas interferon gamma mRNA was detected in 30% of the LL cases. In conclusion, the results obtained in our study confirm and extend earlier observations which examined cytokines in peripheral blood cells and dermal lesions of leprosy. The simplicity of this method would allow the examination of a large number of samples across the spectrum of leprosy.
Shabaana
A K
AK
Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India 625 021.
Venkatasubramani
R
R
Narayan
N S
NS
Hoessli
D C
DC
Dharmalingam
K
K
D001706:Biopsy; D016207:Cytokines / Q000032:analysis / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005260:Female; D006801:Humans; D015440:Leprosy, Lepromatous / Q000276:immunology / Q000378:metabolism* / Q000473:pathology*; D008297:Male; D009166:Mycobacterium leprae / Q000235:genetics / Q000276:immunology*; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016207:Cytokines; D018076:DNA, Complementary; D012333:RNA, Messenger