Full-length poly(A) and mRNA sequencing (FLAM-seq)

We developed FLAM-seq, a fast and simple method for generating cDNA libraries of full-length mRNAs, including the poly(A) tail. By combining a new strategy for cDNA preparation with PacBio long-read sequencing, FLAM-seq enables to generate hundreds of thousands of reads per sample in an easy and short procedure, with starting material ranging from 500 ng to 10 μg of total RNA. Besides the quantification of gene expression and the information about the mRNA isoform, the data generated with FLAM-seq allow accurate measurement of poly(A) tail length for thousands of genes in the sequenced samples. Here we describe the protocol step by step from the RNA preparation to the first steps of data analysis.

The translocation (6;9) (p23;q34) shows consistent rearrangement of two genes and defines a myeloproliferative disorder with specific clinical features

Translocation (6;9)(p23;q34) is a cytogenetic aberration that can be found in specific subtypes of both acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). This translocation is associated with an unfavourable prognosis. Recently, the genes involved in the t(6;9) were isolated and characterized. Breakpoints in both the dek gene on chromosome 6 and the can gene on chromosome 9 appear to occur in defined regions, which allows us to diagnose this type of leukemia at the molecular level. Moreover, because of the translocation a chimeric dek-can mRNA is formed which, as we show here, is an additional target for diagnosis via cDNA-preparation and the polymerase chain reaction (PCR). We studied 17 patients whose blood cells and/or bone marrow cells showed a t(6;9) with karyotypic analysis. Fourteen patients suffered from AML, one patient had a refractory anemia with excess of blasts in transformation (RAEBt), one patient had an acute myelofibrosis (AMF), and one patient a chronic myeloid leukemia (CML). In nine cases studies at the DNA and RNA levels were possible while in seven cases only the DNA could be analyzed. In one case only RNA was available. Conventional Southern blot analysis showed the presence of rearrangements of both the dek gene and the can gene. In both genes, breakpoints cluster in one intron in the patients investigated. The presence of a consistent chimeric dek-can product after cDNA preparation followed by the PCR was demonstrated. We conclude from our data that the t(6;9) is found in myeloproliferative disorders with typical clinical characteristics. This translocation results in highly consistent abnormalities at the molecular level.

The translocation (6;9) (p23;q34) shows consistent rearrangement of two genes and defines a myeloproliferative disorder with specific clinical features

Translocation (6;9)(p23;q34) is a cytogenetic aberration that can be found in specific subtypes of both acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). This translocation is associated with an unfavourable prognosis. Recently, the genes involved in the t(6;9) were isolated and characterized. Breakpoints in both the dek gene on chromosome 6 and the can gene on chromosome 9 appear to occur in defined regions, which allows us to diagnose this type of leukemia at the molecular level. Moreover, because of the translocation a chimeric dek-can mRNA is formed which, as we show here, is an additional target for diagnosis via cDNA-preparation and the polymerase chain reaction (PCR). We studied 17 patients whose blood cells and/or bone marrow cells showed a t(6;9) with karyotypic analysis. Fourteen patients suffered from AML, one patient had a refractory anemia with excess of blasts in transformation (RAEBt), one patient had an acute myelofibrosis (AMF), and one patient a chronic myeloid leukemia (CML). In nine cases studies at the DNA and RNA levels were possible while in seven cases only the DNA could be analyzed. In one case only RNA was available. Conventional Southern blot analysis showed the presence of rearrangements of both the dek gene and the can gene. In both genes, breakpoints cluster in one intron in the patients investigated. The presence of a consistent chimeric dek-can product after cDNA preparation followed by the PCR was demonstrated. We conclude from our data that the t(6;9) is found in myeloproliferative disorders with typical clinical characteristics. This translocation results in highly consistent abnormalities at the molecular level.

Comparison of the levels of the 21S mitochondrial rRNA in derepressed and glucose-repressed Saccharomyces cerevisiae

A cDNA preparation, synthesized by using Saccharomyces cerevisiae mitochondrial RNA as template and oligodeoxythymidylic acid as primer, was found to specifically hybridize to the mitochondrial 21S rRNA by the following criteria: (i) it hybridizes only to the 21S RNA species in mitochondrial RNA and not to RNA from a [rho0] mutant, and (ii) it hybridizes to fragments in restriction digests of mitochondrial DNA that contain the 21S rRNA gene but not to nuclear DNA. This cDNA was used as a probe to demonstrate that a 2.6-fold decrease in the cellular level of the mitochondrial large rRNA is associated with glucose repression of mitochondrial function in S. cerevisiae. A corresponding decrease in the level of mitochondrial DNA was not observed.