Comparison of RNA extraction kits and histological stains for laser capture microdissected prostate tissue

Background Laser capture microdissection offers unique possibilities for the isolation of specific cell populations or histological structures. However, isolation of RNA from microdissected tissue is challenging due to degradation and minimal yield of RNA during laser capture microdissection (LCM). Our aim was to optimize the isolation of high-quality RNA from laser capture microdissected fresh frozen prostate tissue on the level of staining and RNA extraction. Results Cresyl violet and haematoxylin were compared as histological stains for LCM. While RNA quality was similar for cresyl violet (median RIN 7.4) and haematoxylin (median RIN 7.6), tissue morphology was more detailed with cresyl violet as compared to haematoxylin. RNA quality from the following kits was compared: RNeasy® Micro (median RIN 7.2), miRNeasy Mini (median RIN 6.6), Picopure® (median RIN 6.0), mirVana™ miRNA (median RIN 6.5) and RNAqueous®-Micro (median RIN 6.3). RNA quality from microdissected samples with either the RNeasy Micro or miRNeasy Mini kit, was comparable to RNA isolated directly from whole tissue slices (median RIN 7.5, p = 0.09). Isolated RNA from benign and prostate cancer microdissected tissue demonstrated that RNA quality can vary between regions from the same clinical sample. Additionally, RNA quality (r = 0.89), but not quantity (r = 0.69) could be precisely measured with the Agilent Bioanalyzer. Conclusions We demonstrate that staining with cresyl violet results in the isolation of high quality RNA from laser capture microdissected tissue with high discriminative morphology. The RNeasy Micro and miRNeasy Mini RNA extraction kits generated the highest quality RNA compared to Picopure, mirVana and RNAqueous with minimal loss of RNA quality during LCM

18世紀英国の金融不祥事と会計監査 : 「南海の泡沫」(1720)とスネルの「監査報告書」

Epithelial-to-mesenchymal transition (EMT) is a process known to be associated with aggressive tumor behavior, metastasis and treatment resistance. It is characterized by coincidental upregulation of mesenchymal markers such as vimentin, fibronectin and N-cadherin concurrent with E-cadherin downregulation. Studies on EMT are generally perform

Comparison of RNA extraction kits and histological stains for laser capture microdissected prostate tissue

BACKGROUND: Laser capture microdissection offers unique possibilities for the isolation of specific cell populations or histological structures. However, isolation of RNA from microdissected tissue is challenging due to degradation and minimal yield of RNA during laser capture microdissection (LCM). Our aim was to optimize the isolation of high-quality RNA from laser capture microdissected fresh frozen prostate tissue on the level of staining and RNA extraction. RESULTS: Cresyl violet and haematoxylin were compared as histological stains for LCM. While RNA quality was similar for cresyl violet (median RIN 7.4) and haematoxylin (median RIN 7.6), tissue morphology was more detailed with cresyl violet as compared to haematoxylin. RNA quality from the following kits was compared: RNeasy(®) Micro (median RIN 7.2), miRNeasy Mini (median RIN 6.6), Picopure(®) (median RIN 6.0), mirVana™ miRNA (median RIN 6.5) and RNAqueous(®)-Micro (median RIN 6.3). RNA quality from microdissected samples with either the RNeasy Micro or miRNeasy Mini kit, was comparable to RNA isolated directly from whole tissue slices (median RIN 7.5, p = 0.09). Isolated RNA from benign and prostate cancer microdissected tissue demonstrated that RNA quality can vary between regions from the same clinical sample. Additionally, RNA quality (r = 0.89), but not quantity (r = 0.69) could be precisely measured with the Agilent Bioanalyzer. CONCLUSIONS: We demonstrate that staining with cresyl violet results in the isolation of high quality RNA from laser capture microdissected tissue with high discriminative morphology. The RNeasy Micro and miRNeasy Mini RNA extraction kits generated the highest quality RNA compared to Picopure, mirVana and RNAqueous with minimal loss of RNA quality during LCM. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13104-015-1813-5) contains supplementary material, which is available to authorized users

Morphological and immunohistochemical identification of epithelial-to-mesenchymal transition in clinical prostate cancer

Epithelial-to-mesenchymal transition (EMT) is a process known to be associated with aggressive tumor behavior, metastasis and treatment resistance. It is characterized by coincidental upregulation of mesenchymal markers such as vimentin, fibronectin and N-cadherin concurrent with E-cadherin downregulation. Studies on EMT are generally performed in cell lines and mouse models, while the histopathological and phenotypical properties in clinical prostate cancer (PCa) are still unclear. The objective of this study was to identify EMT in PCa patients. We demonstrated that N-cadherin, vimentin and fibronectin were generally not co-expressed in corresponding tumor regions. Immunofluorescent double stainings confirmed that co-expression of mesenchymal markers was uncommon, as we found no prostate cancer cells that co-expressed N-cadherin with fibronectin and only rare (<1%) cells that co-expressed N-cadherin with vimentin. Downregulation of E-cadherin was demonstrated in all N-cadherin positive tumor cells, but not in vimentin or fibronectin positive tumor cells. We further analyzed N-cadherin expression in morphologically distinct PCa growth patterns in a radical prostatectomy cohort (n = 77) and found that N-cadherin is preferentially expressed in ill-defined Gleason grade 4 PCa. In conclusion, we demonstrate that N-cadherin is the most reliable marker for EMT in clinical PCa and is preferentially expressed in ill-defined Gleason grade 4 growth pattern

MET expression during prostate cancer progression

Tyrosine-kinase inhibitors of the hepatocyte growth factor receptor MET are under investigation for the treatment of hormone-refractory prostate cancer (HRPC) metastasis. Analysis of MET protein expression and genetic alterations might contribute to therapeutic stratification of prostate cancer patients. Our objective was to investigate MET on protein, DNA and RNA level in clinical prostate cancer at various stages of progression. Expression of MET was analyzed in hormone-naive primary prostate cancers (N=481), lymph node (N=40) and bone (N=8) metastases, as well as HRPC (N=54) and bone metastases (N=15). MET protein expression was analyzed by immunohistochemistry (D1C2 C-terminal antibody). MET mRNA levels and MET DNA copy numbers were determined by in situ hybridization. None of the hormone-naive primary prostate cancer or lymph node metastases demonstrated MET protein or mRNA expression. In contrast, MET protein was expressed in 12/52 (23%) evaluable HRPC resections. RNA in situ demonstrated cytoplasmic signals in 14/54 (26%) of the HRPC patients, and was associated with MET protein expression (p=0.025, chi(2)), in absence of MET amplification or polysomy. MET protein expression was present in 7/8 (88%) hormone-naive and 10/15 (67%) HRPC bone metastases, without association of HRPC (p=0.37; chi(2)), with MET polysomy in 8/13 (61%) evaluable cases. In conclusion, MET was almost exclusively expressed in HRPC and prostate cancer bone metastasis, but was not related to MET amplification or polysomy. Evaluation of MET status could be relevant for therapeutic stratification of late stage prostate cancer

Differential Expression of the Middle East Respiratory Syndrome Coronavirus Receptor in the Upper Respiratory Tracts of Humans and Dromedary Camels

Middle East respiratory syndrome coronavirus (MERS-CoV) is not efficiently transmitted between humans, but it is highly prevalent in dromedary camels. Here we report that the MERS-CoV receptor-dipeptidyl peptidase 4 (DPP4)-is expressed in the upper respiratory tract epithelium of camels but not in that of humans. Lack of DPP4 expression may be the primary cause of limited MERS-CoV replication in the human upper respiratory tract and hence restrict transmission

Differential expression of the MERS-coronavirus receptor in the upper respiratory tract of humans and dromedary camels

Middle East respiratory syndrome coronavirus (MERS-CoV) is not efficiently transmitted between humans, but it is highly prevalent in dromedary camels. Here we report that the MERS-CoV receptor - dipeptidyl peptidase 4 (DPP4) - is expressed in the upper respiratory tract epithelium of camels but not humans. Lack of DPP4 expression may be the primary cause of limited MERS-CoV replication in the human upper respiratory tract, hence restrict transmission

Differential expression of the Middle East respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels

Middle East respiratory syndrome coronavirus (MERS-CoV) is not efficiently transmitted between humans, but it is highly prevalent in dromedary camels. Here we report that the MERS-CoV receptor-dipeptidyl peptidase 4 (DPP4)-is expressed in the upper respiratory tract epithelium of camels but not in that of humans. Lack of DPP4 expression may be the primary cause of limited MERS-CoV replication in the human upper respiratory tract and hence restrict transmission