Materials and Reagents

1. Total RNA (200 ng~1 ng)
   Note: We have been using 50~200 ng RNA as the starting materials. To compare the results between experiments, it is recommended to use same amount of RNA in all experiments.
   It is recommended to use a method that can purify small RNAs [e.g., TRIzol (Invitrogen, catalog number: 15596-018 ) or miRNeasy Mini Kit (QIAGEN, catalog number: 217004 ). Check the purity of RNA by a spectrophotometer. For consistency of experiments, always use RNA with an A₂₆₀/A₂₈₀ ratio > 1.8.
   Note: Currently, TRIzol is from “Thermo Fisher Scientific, Ambion^TM, catalog number: 15596-018”.
   
2. PCR grade distilled water
   
3. Microtubes for PCR and real-time PCR (MicroAmp^® Optical 8-Cap Strip) (Thermo Fisher Scientific, Applied Biosystems^®, catalog number: 4323032 )
   
4. DNase I (10x buffer is provided along with the enzyme) (Roche Diagnostics, catalog number: 04716728001 )
   
5. Reverse transcription reagent (Transcriptor First Strand cDNA Synthesis kit) (Roche Diagnostics, catalog number: 04897030001 )
   Critical Point: It is recommended to use Transcriptor First Strand cDNA Synthesis kit for measurement of ms² modification. Compared with other reverse transcription kits, this kit gives the highest dynamic range.
   
6. Real-time PCR reagent (SYBR Premix Ex Taq II) (TAKARA BIO INC., catalog number: RR820S )
   Note: SYBR green based PCR reagents from other companies work equally well.
   
7. Primers for detecting mitochondrial ms² modifications in human RNA samples
   
   1. Hu_tRNA^Phe primer f1: CTCCTCAAAGCAATACACTG
      
   2. Hu_tRNA^Phe primer r1: AGCCCGTCTAAACATTTTCA
      
   3. Hu_tRNA^Phe primer r2: GGGTGATGTGAGCCCGTCTA
      
   4. Hu_tRNA^SerUCN primer f1: GAGGCCATGGGGTTGG
      
   5. Hu_tRNA^SerUCN primer r1: CCCAAAGCTGGTTTCAAGC
      
   6. Hu_tRNA^SerUCN primer r2: AATCGAACCCCCCAAAGC
      
   7. Hu_tRNA^Trp primer f1: GGTTAAATACAGACCAAGAGC
      
   8. Hu_tRNA^Trp primer r1: CAACTTACTGAGGGCTTTGAA
      
   9. Hu_tRNA^Trp primer r2: TTAAGTATTGCAACTTACTGAGG
      
   10. Hu_tRNA^Tyr primer f1: GCTGAGTGAAGCATTGGACT
       
   11. Hu_tRNA^Tyr primer r1: AACCCCTGTCTTTAGATTTACA
       
   12. Hu_tRNA^Tyr primer r2: AGAGGCCTAACCCCTGTCTT
8. Primers for detecting mitochondrial ms² modification in mouse RNA samples
   
   1. Ms_tRNAPhe primer f1: GCTTAATAACAAAGCAAAGCA
      
   2. Ms_tRNAPhe primer r1: TATCCATCTAAGCATTTTCA
      
   3. Ms_tRNAPhe primer r2: TGGGATACAATTATCCATCT
      
   4. Ms_tRNA^SerUCN primer f1: CATATAGGATATGAGATTGGC
      
   5. Ms_tRNA^SerUCN primer r1: AACCCCCTAAAATTGGTTTCA
      
   6. Ms_tRNA^SerUCN primer r2: GAAGGAATCGAACCCCCTAA
      
   7. Ms_tRNA^Trp primer f1: GGATATACTAGTCCGCGAGC
      
   8. Ms_tRNA^Trp primer r1: GTGTTTTCTTAGGGCTTTGA
      
   9. Ms_tRNA^Trp primer r2: GTTAAACTTGTGTGTTTTCTTAG
      
   10. Ms_tRNA^Tyr primer f1: ATGGCTGAGTAAGCATTAGA
       
   11. Ms_tRNA^Tyr primer r1: ACCTCTGTGTTTAGATTTAC
       
   12. Ms_tRNA^Tyr primer r2: GAGGATTTAAACCTCTGTGT
       Notes:
       1. Standard PCR grade primers are sufficient for this protocol.
       2. The r1 primer is used for the measurement of total tRNA level, and the r2 primer is
          used for the measurement of ms²-modification level in individual tRNA.

Equipment

1. Conventional PCR apparatus (Thermo Fisher Scientific, Applied Biosystems^®, model: Veriti 96 Well Thermal Cycler )
   
2. Real-time PCR apparatus (Life Technologies, Applied Biosystems^®, model: 7300 Real Time PCR System )
   Note: Currently, it is “Thermo Fisher Scientific, model: 7300 Real Time PCR System ”.
   

Procedure