Researchers at the University of Freiburg aimed to compare the potential of standard RNA-sequencing (RNA-Seq) and 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describe transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. The investigators noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. The researchers thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives.
Comparison between MACE and standard RNA-Seq
a Schematic overview of the two sequencing methods analyzed in this study. In the MACE analysis, only the fragments containing the 3′ end are tagged with a unique molecule identifier, amplified, and sequenced. Consequently, every single read represents a different transcript molecule (left-hand side panel). In the standard RNA-Seq procedure, RNA is fragmented and every fragment is being sequenced (right-hand side panel). b Venn diagram showing the proportion of genes found in common between MACE and standard RNA-Seq analysis (definition of detected genes: mean of normalized reads ≥1 in both groups). c–f Bar plots showing the total number of genes (c), the number of genes with <10 copies (d), ≥10 and <100 copies (e) and ≥100 copies (f) detected by MACE and standard RNA-Seq in unfixed healthy conjunctival tissue. ns not significant.