guinea pig kidney genomic dna library lambda fix ii  (Agilent technologies)

Journal: The Journal of biological chemistry

Article Title: Human Pancreatic Islets Express mRNA Species Encoding Two Distinct Catalytically Active Isoforms of Group VI Phospholipase A 2 (iPLA 2 ) That Arise from an Exon-skipping Mechanism of Alternative Splicing of the Transcript from the iPLA 2 Gene on Chromosome 22q13.1 *

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Figure Lengend Snippet: RT-PCRs were performed with RNA isolated from human islets from two different donors (A) or from two different preparations of human promonocytic U937 cell RNA (B). In A, experiments shown in lanes 1, 2, and 5 were performed with RNA from islets from donor 1, and experiments shown in lanes 3, 4, and 6 were performed with RNA from islets from donor 2. Reverse transcriptase was omitted from the reactions analyzed in lanes 1 and 3 to exclude contamination from genomic DNA in the human islet RNA preparations. In reactions analyzed in lanes 1–4 (A), a set of PCR primers was used that was expected to yield a single 1.65-kb product, based on the rat islet iPLA2 cDNA sequence. In reactions analyzed in lanes 5 and 6 (A), the same 5′-primer was used as in the reactions analyzed in lanes 1–4, but a different 3′-primer was used that was expected to yield a shorter product, based on the rat iPLA2 cDNA sequence. The sequences of the 5′-primer and of the two 3′-primers used in these reactions are specified under “Experimental Procedures.” In B, experiments shown in lanes 1 and 2 were performed with RNA from U936 cell preparation 1, and experiments shown in lanes 3 and 4 were performed with RNA from U937 cell preparation 2. Reverse transcriptase was omitted from the reactions analyzed in lanes 1 and 3 (B). In reactions analyzed in lanes 1–4 (B), the set of PCR primers was the same as that in lanes 1–4 of A. Both of the RT-PCR products visualized in lanes 2 and 4 (B) were subcloned and sequenced, and the results were identical to those for the products in lanes 2 and 4 of A.

Article Snippet: Cloning Human iPLA 2 Genomic DNA Fragments, Determination of Intron-Exon Boundaries, and Estimation of Intron Size A 32 P-labeled human islet iPLA 2 cDNA was used to screen a human placental Lambda FIX II genomic DNA library (Stratagene).

Techniques: Isolation, Sequencing, Reverse Transcription Polymerase Chain Reaction