Journal: Nucleic Acids Research
Article Title: Transcriptome-wide discovery of circular RNAs in Archaea
Figure Lengend Snippet: Experimental verification of cRNAs by RT-PCR. ( A ) Left, RT–PCR results of amplification with outward-directed primers, designed to amplify cRNA; right, RT–PCR results of amplification with inward-directed primers, expected to amplify both linear and cRNA form. Single and multiple arrowheads represent single or double/triple size products, respectively. N/S, non-specific amplification (as verified by direct sequencing). RT–PCR with each primer set was performed on total RNA sample, RNase R-treated sample, and DNA sample, all extracted from S. solfataricus grown to stationary phase on organotrophic medium. ( B ) RT–PCR for verification of circular RNAs. Arrows indicate outward facing primers (top) and inward facing primers (bottom). Purple line/circle denotes the RNA template, pink line denotes expected PCR product. ( C ) Double and triple sized products can stem from multiple rounds of RT around a circular RNA template, followed by PCR amplification. Arrows mark illustrative PCR primers. ( D ) Northern blot analyses of two ncRNAs with circular forms: (M) Size marker, ( 1 ) ncRNA found in genomic location 1 275 500–1 275 567, ( 2 ) ncRNA found in genomic location 442 786–442 854. Circular forms are indicated by ‘C’, and linear forms by ‘L’.
Article Snippet: Total RNA and genomic DNA isolation Total RNA and DNA samples were isolated from each archaeal cells sample (∼107 cells) using TRI reagent according to the manufacturer's protocol (Molecular Research Center Inc.).
Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Polymerase Chain Reaction, Northern Blot, Marker