Journal: The American Journal of Pathology

Article Title: Molecular Characterization of NF-HEV, a Nuclear Factor Preferentially Expressed in Human High Endothelial Venules

doi: 10.1016/s0002-9440(10)63631-0

Figure Lengend Snippet: Figure 4. Virtual Northern and Western blot analyses demonstrating pref- erential expression of NF-HEV in HEVECs. A: Virtual Northern blot analysis of NF-HEV expression in HEVECs, PMECs, HUVECs, or placenta tissue. PCR-generated full-length cDNAs from the various types of ECs were elec- trophoresed on a 1% agarose gel, transferred to nylon filters, and hybridized under high-stringency conditions with a 32P-labeled human NF-HEV cDNA probe. B: Western blot analysis of extracts of tonsillar stroma, HEVECs, PMECs, or HUVECs with rabbit antibodies to NF-HEV. A single band of 30 kd was detected in extracts of tonsillar stroma and HEVECs.

Article Snippet: To obtain sufficient amounts of double-stranded (ds) cDNA for subtraction, both PMEC and HEVEC cDNAs were preamplified with the SMART PCR cDNA Synthesis kit (Clontech, Palo Alto, CA). cDNAs synthesized from 1 g of total RNA (HEVECs) or 0.15 g mRNA (PMECs) with Advantage KlenTaq polymerase (22 cycles, Clontech) were used with the PCR Select cDNA Subtraction kit (Clontech).

Techniques: Northern Blot, Western Blot, Expressing, Generated, Agarose Gel Electrophoresis, Labeling

Journal: Scientific Reports

Article Title: Assessment of Circulating LncRNAs Under Physiologic and Pathologic Conditions in Humans Reveals Potential Limitations as Biomarkers

doi: 10.1038/srep36596

Figure Lengend Snippet: Heatmap showing color coded relative lncRNA plasma levels expressed fold to the PCR detection limit (Cq = 35; Relative Level = 1) after log transformation. Each column represents a unique subject and each row represents a different lncRNA, which have been arranged arbitrarily in ascending (mean) abundance. LncRNAs that were not detected in any subjects are listed next to the heatmap. Right graph shows relative lncRNA levels of individual subjects (circles).

Article Snippet: For assessment of circulating lncRNAs, a fixed volume of total RNA extracted from plasma was input into subsequent cDNA reactions (8 uL/rxn), using the RT 2 lncRNA PreAmp cDNA synthesis kit according to the manufacturer’s instructions (Qiagen).

Techniques: Clinical Proteomics, Transformation Assay

Journal: Scientific Reports

Article Title: Assessment of Circulating LncRNAs Under Physiologic and Pathologic Conditions in Humans Reveals Potential Limitations as Biomarkers

doi: 10.1038/srep36596

Figure Lengend Snippet: ( a ) Heatmap showing color coded relative expression levels of the 84 lncRNAs in normal lung tissue specimens from 4 human donors (without preamplification). Each column represents a unique subject and each row represents a different lncRNA, which have been arranged arbitrarily in ascending (mean) abundance. ( b ) The levels of lncRNA and other small RNA species in lung tissue show a small but statistically significant correlation with their mean plasma levels (n = 4 lung samples; n = 8 plasma samples). Spearman correlation coefficient is shown.

Article Snippet: For assessment of circulating lncRNAs, a fixed volume of total RNA extracted from plasma was input into subsequent cDNA reactions (8 uL/rxn), using the RT 2 lncRNA PreAmp cDNA synthesis kit according to the manufacturer’s instructions (Qiagen).

Techniques: Expressing, Clinical Proteomics

Journal: Scientific Reports

Article Title: Assessment of Circulating LncRNAs Under Physiologic and Pathologic Conditions in Humans Reveals Potential Limitations as Biomarkers

doi: 10.1038/srep36596

Figure Lengend Snippet: ( a ) Heatmap showing of relative expression levels of the 84 lncRNAs in patients with coronary artery disease (CAD), acute coronary syndrome (ACS) or septic shock (SS) (n = 4 patients/group). Color coded relative lncRNA levels are expressed fold to the PCR detection limit after log transformation. Each column represents a unique subject and each row represents a different lncRNA, which have been arranged arbitrarily in ascending (mean) abundance. ( b ) Relative levels of other endogenous plasma RNA species that were extracted and measured in parallel using the same pre-amplification and RT-PCR platform as the lncRNAs. ( c ) Relative levels of several representative endogenous miRNAs and an exogenous spiked-in miRNA mimic (cel-miR-39) that were detected without pre-amplification from the same extracted total RNA. ( d ) Internal reverse transcription (RTC) and positive PCR (PPC) controls were quantified in parallel with the lncRNAs. Cq values for the RTC and PPC of each subject were below manufacturer guidelines (dotted lines), indicative of efficient reverse transcription and PCR reactions.

Article Snippet: For assessment of circulating lncRNAs, a fixed volume of total RNA extracted from plasma was input into subsequent cDNA reactions (8 uL/rxn), using the RT 2 lncRNA PreAmp cDNA synthesis kit according to the manufacturer’s instructions (Qiagen).

Techniques: Expressing, Transformation Assay, Clinical Proteomics, Amplification, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription

Journal: Scientific Reports

Article Title: Assessment of Circulating LncRNAs Under Physiologic and Pathologic Conditions in Humans Reveals Potential Limitations as Biomarkers

doi: 10.1038/srep36596

Figure Lengend Snippet: ( a ) Heatmaps showing relative levels of Lipcar (L), Tapsaki (T) and Coromarker (C) in plasma from five different groups of subjects or patients, and normal lung tissue specimens. Color coded relative lncRNA levels are expressed fold to the PCR detection limit after log transformation. Each row represents a different subject or patient. Plasma lncRNA levels were quantified after pre-amplification (+PreAmp), while lung levels were quantified without the need for pre-amplification (−PreAmp). ( b ) No significant differences in plasma Lipcar levels observed between groups (Kruskal-Wallis non-parametric test and Dunn’s multiple comparison test). Lipcar levels were normalized to B2M.

Article Snippet: For assessment of circulating lncRNAs, a fixed volume of total RNA extracted from plasma was input into subsequent cDNA reactions (8 uL/rxn), using the RT 2 lncRNA PreAmp cDNA synthesis kit according to the manufacturer’s instructions (Qiagen).

Techniques: Clinical Proteomics, Transformation Assay, Amplification, Comparison