Application of RT-PCR in formalin-fixed and paraffin-embedded lung cancer tissues.
OBJECTIVE
To analyze gene expression in formalin-fixed, paraffin-embedded lung cancer tissues using modified method.


METHODS
Total RNA from frozen tissues was extracted using TRIZOL reagent. RNA was extracted from formalin-fixed, paraffin-embedded tissues by digestion with proteinase K before the acid-phenol:chloroform extraction and carrier precipitation. We modified this method by using a higher concentration of proteinase K and a longer digestion time, optimized to 16 hours. RT-PCR and real-time RT-PCR were used to check reproducibility and the concordance between frozen and paraffin-embedded samples.


RESULTS
The results showed that the RNA extracted from the paraffin-embedded lung tissues had high quality with the most fragment length between 28S and 18S bands (about 1000 to 2000 bases). The housekeeping gene GUSB exhibited low variation of expression in frozen and paraffin-embedded lung tissues, whereas PGK1 had the lowest variation in lymphoma tissues. Furthermore, real-time PCR analysis of the expression of known prognostic genes in non-small cell lung carcinoma (NSCLC) demonstrated an extremely high correlation (r>0.880) between the paired frozen and formalin-fixed, paraffin-embedded specimens.


CONCLUSIONS
This improved method of RNA extraction is suitable for real-time quantitative RT-PCR, and may be used for global gene expression profiling of paraffin-embedded tissues.
Zhang
Fan
F
Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment; Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin, China.
Wang
Zhuo-min
ZM
Liu
Hong-yu
HY
Bai
Yun
Y
Wei
Sen
S
Li
Ying
Y
Wang
Min
M
Chen
Jun
J
Zhou
Qing-hua
QH
D000230:Adenocarcinoma / Q000235:genetics; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000235:genetics; D005557:Formaldehyde / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D005966:Glucuronidase / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D016612:Paraffin Embedding / Q000379:methods; D010735:Phosphoglycerate Kinase / Q000235:genetics; D012313:RNA / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D005557:Formaldehyde; D012313:RNA; D010735:Phosphoglycerate Kinase; D005966:Glucuronidase