Journal: Genome Research

Article Title: 5?-Transducing SVA retrotransposon groups spread efficiently throughout the human genome

doi: 10.1101/gr.093435.109

Figure Lengend Snippet: Ongoing transcription of SVA_F1 subfamily members is demonstrated by ESTs and RT-PCR analyses. (A) ESTs (arrows) aligning specifically to the source element H10_1 and its descendants (DB277120, AA582071) and to MAST2–SVA junctions of a subset of SVA_F1 subfamily members. Extensions of the queries used to identify ESTs (black bars) are shown. (B) Transcriptional control of transduction group 1 members H20_1 and H1_F_160 is effected by external promoters. Identified ESTs match 5′ junctions of the SVA elements with primary 5′ transductions. MER57A is an internal portion of the nonautonomous retrovirus 1 (ERV1). (C) ESTs indicating ongoing transcription of source element HX_4. Those ESTs that correspond to the 3′-end (BI325030, AW627827) and the 3′ junction (BX110444) of HX_4 are also consistent with transcription of the HX_4 derivative H3_8 (Fig. 7C). 3′-Ends of sense ESTs BI325030 and BX110444 as well as the 5′-end of antisense EST BX373748 are directly at the polyadenylation sites of HX_4 and/or H3_8. SVA copies highlighted in red were verified to be source elements. (D) RT-PCR analyses confirm transcription of distinct SVA_F1 subfamily members. Agarose gel electrophoresis of H20_1 and HX_4/H3_8-specific RT-PCR products derived from total RNA from human tissues and NCCIT cells. Primer binding sites and extension of the RT-PCR products are indicated as short arrows and red bars, respectively, in B and C. To control for both successful cDNA synthesis and DNA-free RNA preparations, PCRs with GAPDH-specific, intron-spanning primers (see Methods) were performed on RNA preparations from each tissue before (−RT) and after (+RT) cDNA synthesis. PCR on genomic HeLa DNA served as a positive control. The 766- and 481-bp products originate from the unspliced GAPDH gene and a processed GAPDH pseudogene, respectively (Harper et al. 2003). Sizes of obtained PCR products are indicated and correspond to expected fragment sizes. RT, reverse transcriptase; M, size marker; NTC, no template control.

Article Snippet: Total RNA was extracted from human teratocarcinoma cell line NCCIT (ATCC CRL-2073) using TRIzol reagent (Invitrogen).

Techniques: Reverse Transcription Polymerase Chain Reaction, Transduction, Agarose Gel Electrophoresis, Derivative Assay, Binding Assay, Positive Control, Marker

Journal: Frontiers in Immunology

Article Title: Immune Response to Enterococcus gallinarum in Lupus Patients Is Associated With a Subset of Lupus-Associated Autoantibodies

doi: 10.3389/fimmu.2021.635072

Figure Lengend Snippet: Anti-human RNA IgG antibodies in anti-Ribosomal P (A) and anti-dsDNA (C) negative and positive patients. All samples were tested at a 1:100 serum dilution and results are shown as absorbance at 450nm. Antibody levels were compared using non-parametric Mann-Whitney test. Number of samples are shown in parentheses. Correlation between anti-human RNA IgG and anti- Eg IgG titers in patients positive for anti-Ribosomal P (B) and anti-dsDNA (D) . OD, optical density.

Article Snippet: Human RNA was purified from THP1 (ATCC#TIB-202), a human monocytic cell line, propagated in RPMI-1640 with 10% bovine calf serum.

Techniques: MANN-WHITNEY

Journal: BMC Molecular Biology

Article Title: miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample

doi: 10.1186/1471-2199-9-34

Figure Lengend Snippet: Amplification plot and melting curve of the proposed miR-145 miR-Q assay . A) Amplification plot of synthetic miR-145 with 50 ng bacterial total RNA present as complex background. Target input ranged between 2 nM and 0.2 fM in a miR-Q reaction. B) SYBR green melting curve of the miR-145 assay. Neither the non-spiked bacterial total RNA control, nor H 2 O control showed a background signal over 40 cycles.

Article Snippet: Human total RNA from the cervical carcinoma cell line HeLa (ATCC No.: CCL-2), the colon carcinoma cells HT-29 (ATCC No.: HTB-38), and the lung carcinoma cell line A549 (ATCC No.: CCL-185) as well as porcine total RNA from intestinal tissues were prepared using the mirVana miRNA Isolation Kit (Ambion, Darmstadt, Germany), according to the manufacturer's protocol.

Techniques: Amplification, SYBR Green Assay, Control

Journal: BMC Molecular Biology

Article Title: miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample

doi: 10.1186/1471-2199-9-34

Figure Lengend Snippet: Side-by-side quantification of let-7b in human total RNA samples isolated from the cell lines: A549, HeLa, and HT-29 in two independent qPCR runs . Experiments were performed using the miR-Q approach as well as the mirVana™ qRT-PCR Detection Kit. For this purpose, total RNA samples (50 ng/μl) were either spiked with 100 pM synthetic let-7b (A549 si, HeLa si, and HT-29 si) or remained non-spiked (A549, HeLa, and HT-29). RT reactions were performed with 50 ng, 25 ng, and 5 ng of all RNA samples, followed by qPCR detection of let-7b in different runs. Columns represent the mean (± SD) of three measurements. A) let-7b quantification by means of the miR-Q approach using both the spike-in controls and the non-spiked samples. B) Quantification of let-7b by means of mirVana™ qRT-PCR using both the spike-in controls and the non-spiked samples.

Article Snippet: Human total RNA from the cervical carcinoma cell line HeLa (ATCC No.: CCL-2), the colon carcinoma cells HT-29 (ATCC No.: HTB-38), and the lung carcinoma cell line A549 (ATCC No.: CCL-185) as well as porcine total RNA from intestinal tissues were prepared using the mirVana miRNA Isolation Kit (Ambion, Darmstadt, Germany), according to the manufacturer's protocol.

Techniques: Isolation, Quantitative RT-PCR