Functional Genomics by Michael Kaufmann Claudia Klinger & Andreas Savelsbergh

Author:Michael Kaufmann, Claudia Klinger & Andreas Savelsbergh
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

1.2 Multiplex Sequencing Strategy

Starting from frozen or fresh tissue, cell culture, FFPE or blood samples, total RNA is isolated by using a mixture of acidified phenol/guanidine thiocyanate and chloroform or column-based extraction methods. When having fresh tissue initial enzymatic digestion and cell separating steps using, i.e., magnetic activated cell sorting (such as Miltenyi Biotec’s MACS) should be considered to obtain purified cell types. To assess the quantity of total RNA a Photometer (such as Implen’s NanoPhotometer) can be used to measure the absorbances at 260–280 nm. A ratio of absorbance at 260–280 nm of ~2.0 is indicative of successful RNA purification. If the ratio is appreciably lower, it may indicate the presence of protein, phenol, or other contaminants that absorb strongly around 280 nm. In that case RNA concentration could be overestimated. It could be applicable to use a fluorimeter (such as Life Technologies’ Qubit or Promega’s Quantus) and a Bioanalyzer (such as Agilent’s 2100 Bioanalyzer), because these methods are less sensitive to contaminants such as phenol or genomic DNA. On a fluorimeter the RNA concentration is measured by the absorbance of a fluorescent dye which is bound to the RNA. The Bioanalyzer is a micro-scale electrophoresis as well based on the absorbance of a fluorescent dye bound to the RNA and the different elution times of different sized RNA fragments. The so-called electropherogram of eukaryotic RNAs mainly shows two well-defined peaks corresponding to the 18S ribosomal (rRNA) of 1.9 kb and 28S rRNA of 4.5 kb with a ratio of approximately 2:1. This total RNA ratio is calculated by taking the ratio of the area under the 18S and 28S rRNA peaks to the total area under the graph. The Bioanlayzer software uses algorithms based on the integrity of the electropherogram to calculate the RNA Integrity Number (RIN) with a value of 1–10, with 10 being the highest. The RIN (given by electropherogram) for total RNA input should be >7.

Small RNAs are purified using polyacrylamide gel electrophoresis (PAGE) (see Fig. 1a). A ladder containing two DNA oligonucleotides (18 bases and 36 bases) indicates the area that has to be cut for small RNA purification (see Fig. 1b). The subsequent cDNA library preparation is a four-step process starting with the ligation of a DNA oligonucleotide (3′-adapter) to the 3′-end of the selected RNAs, followed by ligation of a RNA oligonucleotide (5′-adapter) to the 5′-end of the selected RNAs (see Fig. 2). The resulting molecules are transcribed via reverse transcription (RT) and amplified via PCR.

Fig. 1Enrichment of small RNAs is essential in order to obtain high numbers of reads. (a) Selection of small RNAs using polyacrylamide gel electrophoresis. Only the ladder and the first approx. 5 cm of each sample (exhibiting enrichment of larger RNAs, such as t-RNAs, r-RNAs, and mRNA fragments) exposed to UV. (b) Areas containing RNA of 18–36 nucleotides cut from the gel separately for each sample

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