Cracking it - successful mRNA extraction for digital gene expression analysis from decalcified, formalin-fixed and paraffin-embedded bone tissue

With the advance of precision medicine, the availability of tumor tissue for molecular analysis has become a limiting factor. This is particularly the case for bone metastases which are frequently occurring in cancer types such as prostate cancer. Due to the necessary decalcification process it was long thought that transcriptome analysis will not be feasible from decalcified formalin-fixed, paraffin-embedded (DFFPE) in a large manner. Here we demonstrate that mRNA extraction from DFFPE is feasible, quick, robust and reproducible and that decalcification does not hamper subsequent gene expression analysis. This might assist in implementing transcriptome analysis from DFFPE into every day practice.

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Amplification-free gene expression analysis of formalin-fixed paraffin-embedded samples using scanning single-molecule counting

Analytical Biochemistry ◽

10.1016/j.ab.2021.114220 ◽

2021 ◽

Vol 625 ◽

pp. 114220

Author(s):

Hidetaka Nakata ◽

Mitsushiro Yamaguchi ◽

Takuya Hanashi ◽

Seiji Kondo ◽

Tetsuya Tanabe

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Single Molecule ◽

Gene Expression Analysis ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Free Gene ◽

Formalin Fixed

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Maximizing RNA Yield from Archival Renal Tumors and Optimizing Gene Expression Analysis

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109355059 ◽

2009 ◽

Vol 15 (1) ◽

pp. 80-85 ◽

Cited By ~ 10

Author(s):

Sean T. Glenn ◽

Karen L. Head ◽

Bin T. Teh ◽

Kenneth W. Gross ◽

Hyung L. Kim

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Reverse Transcription ◽

Gene Expression Analysis ◽

Rna Isolation ◽

Proteinase K ◽

Specific Primers ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan ® PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPure™ kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen’s TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers’ protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan ® PCR. Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqMan® qPCR can be optimized by using the MasterPure™ RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription.

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