This protocol is used for mapping and discrimination of the primary and processed transcripts in maize mitochondria. This technique includes an RNA normalization step and it minimize the influence of unstable five prime triphosphate that hinders a clear discrimination between the primary and the processed transcripts. Besides maize mitochondria, this method could be applied to plastids and other plant mitochondria.
Start by collecting 20 grams of developing kernels into a 50 milliliter tube on ice. Transfer the kernels to a pre-cooled mortar and add 10 to 20 milliliters of ice cold extraction buffer. Grind the kernels completely, add more extraction buffer and filter the ground tissue through two layers of filter cloth.
Centrifuge the filtrate at 8, 000 times G for 10 minutes. Then transfer the supernatant to a new tube and discard the filtrate. Centrifuge the tube at 20, 000 times G for 10 minutes.
Pour off the supernatant and re-suspend the pellet in six milliliters of wash buffer. Aliquot the suspension into five 1.5 milliliter RNase-free tubes and centrifuge them at 14, 000 times G for five minutes. Discard the supernatant and freeze the mitochondrial pellet in liquid nitrogen.
Extract mitochondrial RNA using commercial reagents according to the manufacturer's instructions and set up an RNA five prime polyphosphaatase treatment. Then, recover the RNA with an RNA purification kit. The reagent used for RNA extraction is hazardous.
Always work with it in a fume hood and always wear lab coat and gloves. Prepare two circularization reactions using the same amounts of five prime polyphosphatase treated and non-treated mitochondrial RNA's. Incubate both reactions at 16 degrees celsius for 12 to 16 hours and then recover the self-ligated RNA's with a previously used RNA purification kit.
Start by preparing a primer mixture with an equal ratio of 26 SCRT and up to seven other reverse transcription primers. Assemble two reverse transcription reaction systems according to the manuscript directions and incubate them at 42 degrees celsius for 50 minutes. To normalize the cDNA, prepare two PCR reactions with the same volume of cDNA's from the five prime polyphosphatase treated or non-treated RNA's and run the reaction under thermocycling conditions described in the manuscript.
Normalization by 26S mature rRNA is a key step to discriminate between the primary and the processed transcripts. Compare the abundance of the two PCR products and if necessary, optimize the normalization by adjusting the amounts of template cDNA's. Prepare pairs of PCR reactions with appropriate volumes of normalized cDNA's and a pair of divergent primers like in the five prime to three prime junction of the target transcripts and perform PCR according to the manuscript directions.
Then, use a gel DNA recovery kit to isolate the prominent bands that are amplified by nested PCR. Clone the gel purified PCR products into a vector using standard techniques and perform colony PCR to select positive clones containing the target inserts. Sequence the PCR products and align the sequencing data with the maize mitochondrial genome using the basic local alignment search tool or BLAST.
Choose the organism maize and search database nucleotide collection. Find the five prime to three prime junction of the circularized transcript and determine the positions of the five prime and three prime transcript termini. Amplify the DNA fragment used to prepare the RNA probe and clone it to the previously used vector which contains a T7 promoter, 17 base pairs upstream of the insertion site.
Label the RNA probes with dig-11-UTP and perform RNA hybridization using commercial kits. Discriminate the primary and processed five prime ends by comparing the circular RT-PCR products obtained from the normalized five prime polyphosphatase treated and non-treated RNA's. Then, design quantitative RT-PCR primers based on the mapping results and verify the discrimination results with RT-PCR.
The COX-2 gene was used as an example to demonstrate the mapping and discrimination of maize mitochondrial transcripts with the circular RT-PCR based strategy. The normalized cDNA's were used as templates for PCR, which resulted in two prominent bands amplified from the five prime polyphosphatase treated sample. The two bands were named COX-2 one and two recovered from the gel and cloned into vectors.
Colony PCR results showed that the positive clones contain inserts with variable size which implies that the termini have heterogeneous five prime or three prime termini. The sequencing results showed that COX-2 one and two have the same three prime ends at 39 nucleotides downstream of the stop codon while their five prime termini are located at 992 to 1, 030 and 1, 276 to 1, 283 nucleotides upstream of the start codon, respectively. RNA gel blot hybridization was performed to verify the circular RT-PCR mapping results and two major bands with sizes similar to COX-2 one and two were detected.
Another pair of divergent primers were designed to amplify the COX-2 three and four bands detected with the RNA gel blot but not with first time circular RT-PCR. The quantitative RT-PCR results confirmed that COX-2 one, two, three and four were sensitive to five prime polyphosphatase and that they had primary five prime termini. Primer extension analysis could be performed to verify the five prime mapping results although it's not included in this protocol.
