Application of RT-PCR in formalin-fixed and paraffin-embedded lung cancer tissues

Abstract

Aim: 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.

Conclusion: 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.

Figure 1

Agarose gel electrophoresis of total RNA extracted from frozen lung cancer tissues (A) and the corresponding paired formalin-fixed, paraffin-embedded lung cancer tissues (B). Lanes 1−4: one-year old samples; Lanes 5−6: 3-year old samples; Lanes 7−8: 5-year old samples.

Figure 2

Effects of the method of total RNA extraction from formalin-fixed, paraffin-embedded lung cancer tissues on amplifiable RNA fragment length. Thirteen primer pairs were tested that amplify from 99 to 705 bp of β-actin. The expected product size is given for amplicons amplified from the frozen (A) and formalin-fixed, paraffin-embedded (B) lung cancer specimens.

Figure 3

RNA expression of housekeeping genes in paired frozen and formalin-fixed, paraffin-embedded lung cancer tissues. (A) Mean RNA expression (represented by mean C_T) of 4 housekeeping genes (GUSB, PGK-1, GAPDH, and 18S) in 8 frozen and paired formalin-fixed, paraffin-embedded lung cancer tissues. Each bar represents gene expression in an individual specimen. Numbers above the bar represent the variance of expression of particular gene among the specimens. (B) RNA expression of PGK-1, GUSB, LCK, STAT1, MMD ERBB3, and DUSP6 in formalin-fixed, paraffin-embedded lung cancer tissues. The expression of each gene is normalized to its expression in the matched frozen specimens, as represented by C_T difference between the paraffin and frozen specimens. Each symbol represents a distinct paired tissue specimen.

Figure 4

Comparison of RT-PCR expression profiles of 5 genes (LCK, MMD, STAT1, ERBB3, and DUSP6) from paired frozen and formalin-fixed, paraffin-embedded lung cancer tissues. (A) Total RNA was extracted from frozen and paraffin-embedded lung cancer tissues, mRNA levels were determined by real-time SYBR green RT-PCR as described in Material and Methods. Each bar represents the normalized expression relative to GUSB and the mean of three measurements: Grey — formalin-fixed, paraffin-embedded tissues; Black — frozen tissues. (B) Spearman correlation for the 5 gene expression in 8 paired frozen and formalin-fixed, paraffin-embedded lung cancer tissues.