Journal: Laboratory Investigation

Article Title: Amplification of RNA transcripts using terminal continuation

doi: 10.1038/sj.labinvest.3700005

Figure Lengend Snippet: Figure 1 Overview and analysis of the TC RNA amplification method. (a) A TC primer (containing a bacteriophage promoter sequence for sense orientation) and a poly-d(T) primer are added to the mRNA population to be amplified (green rippled line). First strand (blue line) synthesis occurs as an mRNA–cDNA hybrid is formed after reverse transcription and terminal continuation of the oligonucleotide primers. Following RNase H digestion to remove the original mRNA template strand, ss (red line) synthesis is performed using Taq polymerase. The resultant ds product is utilized as template for in vitro transcription, yielding high fidelity, linear RNA amplification of sense orientation (green rippled lines). (b) Schematic similar to (a) illustrating the TC RNA amplification procedure amplifying RNA in the antisense orientation (yellow rippled lines). (c)Bioanalysis of TC RNA amplification products to illustrate relative abundance and distribution. Note the long-range representation of transcripts from high (greater than 7.5 kb) to low (100 bp) on the electro- pherogram gel. Lane 1 depicts the molecular weight standards. Lane 2 is a control lane with a commercially available purified mRNA (arrowhead) used for amplification in lanes 3–5. Highly consistent amplification is observed in representative TC RNA amplification reactions shown in triplicate (lanes 3–5). Note the relative paucity of signal variation from the three separate reactions. Amplification efficiency of approximately 2500–3000- fold is estimated using the control poly-A þ mRNA as a starting template.

Article Snippet: Extracted RNAs are obtained from single cells/ populations using microaspiration or LCM techniques.4,8,16 In addition to the microaspirated samples, a 1:20 dilution series (sampling at 1:1, 1:20, 1:400, 1:8000, 1:160 000, and 1:3 200 000) of RNA extracted from mouse hippocampus was performed to assess signal recovery using the same TC RNA amplification procedures.

Techniques: RNA Amplification, Sequencing, Amplification, Reverse Transcription, In Vitro, Molecular Weight, Control, Purification

Journal: Laboratory Investigation

Article Title: Amplification of RNA transcripts using terminal continuation

doi: 10.1038/sj.labinvest.3700005

Figure Lengend Snippet: Figure 2 TC RNA amplification of single cells and discrete regions in the human hippocampus. (a) Bioanalysis illustrating the robust effect of TC RNA amplification. Note the strong signal intensity from a range of high to low molecular weights (47.5 kb to o100 bp), indicating amplification of large and small tran- scripts and the lack of a ‘blob’ of short oligonucleotides at the lower molecular weights that often occurs with conventional aRNA methodologies. Lanes 1 and 2 depict TC RNA amplification from paraffin-embedded hippocampus, whereas lanes 3 and 4 are single-cell amplifications of individual neurofilament-immunor- eactive CA1 pyramidal neurons from the same human brain, microdissected from a 70% ethanol fixed, paraffin-embedded tissue section of the hippocampus by microaspiration. Lane 5 illustrates a p75 (low-affinity nerve growth factor receptor)- immunoreactive cholinergic basal forebrain (CBF) neuron micro- aspirated from a paraformaldehyde fixed, frozen section of the basal forebrain. (b) Two identical custom-designed arrays hybri- dized with 33P-UTP labeled probes generated using TC RNA amplification from adjacent samples from the same case (corre- sponding lanes 1 and 2 of panel (a)) using organic (top panel) and magnetic extraction methods (bottom panel). No quantitative differences have been observed between extraction conditions.

Article Snippet: Extracted RNAs are obtained from single cells/ populations using microaspiration or LCM techniques.4,8,16 In addition to the microaspirated samples, a 1:20 dilution series (sampling at 1:1, 1:20, 1:400, 1:8000, 1:160 000, and 1:3 200 000) of RNA extracted from mouse hippocampus was performed to assess signal recovery using the same TC RNA amplification procedures.

Techniques: RNA Amplification, Amplification, Labeling, Generated, Extraction

Journal: Laboratory Investigation

Article Title: Amplification of RNA transcripts using terminal continuation

doi: 10.1038/sj.labinvest.3700005

Figure Lengend Snippet: Figure 3 Expression profile distribution and linearity following TC RNA amplification. (a) Dendrogram illustrating expression levels for 4230 genes from individual CA1 pyramidal neurons aspirated from the hippocampus of postmortem human brain. TC1a–TC1c represent TC RNA amplifications run in triplicate from adjacent cells. Note the wide range of color-coded relative expression levels and the high degree of similarity of expression profiles for the three samples. (b) Scatter plots demonstrating a linear relationship between TC-amplified RNA input concentra- tion and mean hybridization signal intensity of 96 cDNAs/ESTs on a custom-designed array (r2 ¼ 0.98) as well as for an individual clone (CREB is depicted; r2 ¼ 0.96). (c) Histogram illustrating mean hybridization signal intensity with standard deviations from mouse hippocampus (run three to four times per dilution) to illustrate the high level of signal recovery following serial 20-fold dilutions. Average calculated recoveries from serial dilutions: 1:1; 1:20 (actual recovery 1:26.476.8), 1:400 (actual recovery 1:457.4744.6), 1:8000 (actual recovery 1:5864.27478.3), 1:160 000 (actual recovery 1:126 13971788.6), and 1:3 200 000 (N/D). Note the logarithmic scale and limit of detection at the final dilution.

Article Snippet: Extracted RNAs are obtained from single cells/ populations using microaspiration or LCM techniques.4,8,16 In addition to the microaspirated samples, a 1:20 dilution series (sampling at 1:1, 1:20, 1:400, 1:8000, 1:160 000, and 1:3 200 000) of RNA extracted from mouse hippocampus was performed to assess signal recovery using the same TC RNA amplification procedures.

Techniques: Expressing, RNA Amplification, Amplification, Hybridization

Journal: Laboratory Investigation

Article Title: Amplification of RNA transcripts using terminal continuation

doi: 10.1038/sj.labinvest.3700005

Figure Lengend Snippet: Figure 4 Comparison of TC RNA amplification hybridization signal intensity with conventional RNA amplification. The left panel illustrates a comparison of two adjacent regions from the same hippocampal tissue section amplified by TC RNA amplifi- cation (top panel) and by conventional RNA amplification (bottom panel). The relative hybridization signal intensity of several low, moderate, and higher expressing genes is depicted. All steps in the procedures are performed identically, including hybridization time, identical washing regimens, and source of the custom-designed arrays. Significant increases in gene expression levels are detected (* denotes Po0.05 and **denotes Po0.01) for b-actin (b-act*), b-tubulin (b-tub*), neurofilament subunits (NF- L*, NF-M**, and NF-H**), 4-repeat tau (tau44*), nestin (nest*), utrophin (utro**), AMPA glutamate receptors (GluR1*, GluR3*), and kainite receptors (GluR5**, GluR6**, and GluR7**). Other clones represented on the arrays include: g-actin (g-act), a-tubulin (a-tub), microtubule-associated proteins 2 and 5 (MAP2 and MAP5), largest isoform tau and three-repeat tau (tau40 and tau43), internexin (INX), AMPA glutamate receptors (GluR2 and GluR4), and kainate receptors (KA1 and KA2). The right panel depicts a histogram illustrating mean hybridization signal intensity for custom-designed cDNA arrays (containing 230 cDNAs/ESTs) using 50 TC RNA amplification (sense orientation), 30 TC RNA amplification (antisense orientation), and aRNA amplification from the same hippocampal tissue sections. A total of 12 reactions from three brains (four RNA amplification reactions per brain) are performed for each method and reported as normalized hybridi- zation signal intensity7s.e.m. A 3.5–4-fold increase in signal intensity is observed for both 50 and 30 TC RNA amplification methods (see Table 2 for further details).

Article Snippet: Extracted RNAs are obtained from single cells/ populations using microaspiration or LCM techniques.4,8,16 In addition to the microaspirated samples, a 1:20 dilution series (sampling at 1:1, 1:20, 1:400, 1:8000, 1:160 000, and 1:3 200 000) of RNA extracted from mouse hippocampus was performed to assess signal recovery using the same TC RNA amplification procedures.

Techniques: Comparison, RNA Amplification, Hybridization, Amplification, Expressing, Gene Expression, Clone Assay