Messageamp arna kit

For each sample, RNA was extracted from 30 to 50 sections of isolated tissues using the Absolutely RNA Nanoprep Kit (Stratagene). Total RNA was amplified by one round of T7-based mRNA amplification using the MessageAmp aRNA Kit (Ambion) to generate tissue-specific antisense RNA (aRNA). After quality assessment of aRNA populations first strand cDNA was synthesized using SuperScript III (Invitrogen) with random priming according to the manufactureŕs instructions. The Power SYBR Green PCR master mix was used to perform reactions in an ABI 7900 HT Real-Time PCR system (Applied Biosystems). Data were analyzed using SDS 2.2.1 software (Applied Biosystems). Three replications were conducted for each transcript. The data were analyzed by ANOVA followed by a posthoc-test using Microsoft Excel with Daniel’s XL toolbox version 6.10 [57] .
The highest relative expression in the group of genes was taken to 100% and expression of the other genes and stages was re-calculated to that value. Primers used for qRT-PCR are listed in Table S1.

Because of the small amount of RNA extracted from hair samples, a double RNA amplification step was incorporated prior to microarray hybridization. Total RNA was amplified using the Ambion MessageAmp aRNA Kit as described previously [28 (link)]. Briefly, first- and second-strand cDNA were synthesized. Unlabeled amplified (aRNA) was generated by in vitro transcription with non-biotinylated NTPs. For probe preparation, amplified aRNA was reverse-transcribed with second-round primers. The second-strand cDNA was synthesized with T7 oligo (dT) primer and purified. Biotin-labeled cRNA was generated by in vitro transcription and purified with the RNeasy Kit (Qiagen, Venlo, Netherlands).

Details of the tissue collection and gene expression profiling have been previously described in detail [8] (link). Briefly, transrectal ultrasound-guided needle biopsies were obtained from each patient at study entry and snap-frozen in liquid nitrogen prior to chemotherapy. At radical prostatectomy, cancer-containing tissue samples were snap frozen immediately after prostate removal. Benign and neoplastic epithelial cells were separately acquired by laser-capture microdissection (LCM) using an Arcturus PixCell IIe microscope (Arcturus, Mountain View, CA). Total RNA was extracted from captured epithelium using a Picopure RNA isolation kit according to the manufacturer's instructions (Arcturus Inc., Mountain View, CA), and amplified using the messageAMP aRNA kit (Ambion, Austin, TX). Labeled cDNA probes were hybridized in a head-to-head fashion, pre-chemotherapy versus post-chemotherapy simultaneously to cDNA microarrays as we have previously described [14] (link).

Total RNA was extracted from embryo, endosperm and seed coat of different developmental stages following the protocol provided by the RNAqueous-Micro kit (Ambion catalog number 1927). The quantity of RNA isolated from early-stage embryos was insufficient for library preparation for RNA-seq experiments. Therefore, the mRNA from all stages was amplified and the antisense RNA (aRNA) was used for RNA-seq analysis. The mRNA amplification was conducted according to the protocol provided in the MessageAmp aRNA kit (Ambion catalog number 1750).

The total RNA isolated from short-term spermatogonia and long-term haGSC cultures, hESC line H1 (positive control), and testicular fibroblasts (hFibs; negative control) was prepared using the RNeasy Mini Kit (Qiagen), followed by an amplification step with MessageAmp aRNA Kit (Ambion). In each sample, 200 cells were collected per probe with the micromanipulation system and transferred directly into 10 μL of RNA direct lysis solution and stored at −80°C. Samples were analysed at the microarray facility of the University of Tübingen Hospital, Germany. Gene expression analysis was performed using the Human U133 + 2.0 Genome oligonucleotide array (Affymetrix). The raw data (CEL-files) was provided to the MicroDiscovery GmbH, Berlin, Germany, for normalization and biostatistical analysis.

Total RNA (2 μg) was amplified and biotin-labeled by a round of in vitro transcription with a Message Amp aRNA kit (Ambion, Austin, Texas, USA) following the manufacturer's protocol. Before amplification, spikes of synthetic mRNA at different concentrations were added to all samples; these positive controls were used to ascertain the quality of the process. aRNA yield was measured using a UV spectrophotometer and the quality on nanochips with the Agilent 2100 Bioanalyzer (Agilent).

Total RNA was extracted from lectin-selected Sertoli using the RNeasy Mini Kit (Qiagen), and then amplified using the MessageAmp aRNA Kit (Ambion, Austin, TX, USA). Moreover, 100 hundred cells were harvested per probe in each sample using the micromanipulation equipment, transferred immediately into 10 L of RNA direct lysis solution, and kept at –80 °C. The RNA was isolated from cell samples and microarray datasets obtained from the hybridization of human mRNA-derived cDNA after amplification using Super-Amp^TM technology. Agilent Whole Human Genome Oligo Microarrays 8 × 60 K v² (Miltenyi, Germany) were analyzed by bioinformatics tools.