Measurement of 2-methylthio Modifications in Mitochondrial Transfer RNAs by Reverse-transcription Quantitative PCR   

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

Part I. DNase treatment

1. Prepare total RNA from cell and tissue samples of human or animals by TRIzol reagent or miRNeasy kit according to the manufacturer’s instructions.
   
2. Mix following reagents in microtubes for DNase treatment on ice.
   

   Component	Volume (µl/reaction)
   RNA (100 ng/µl)	2
   DNase I	0.5
   10x DNase I buffer	2
   PCR grade water	15.5
   Total volume	20

   Note: For measurement of ms² modification in multiple samples, it is recommended to prepare a master mixture of all reagents except RNA to reduce variability. When preparing a master mixture, it is usually desirable to prepare a slight excess to compensate for pipetting inaccuracies.
   
3. Put microtubes in PCR apparatus and use the following program.
   

   Time and temperature
   Digestion	Inactivation	Hold
   37 °C for 20 min	75 °C for 10 min	4 °C

   
   
4. Use DNase I-treated sample for subsequent reverse transcription or store at -80 °C.

Part II. Reverse transcription


Figure 1. Workflow of the method to detect ms² modification. The mitochondrial tRNA is reversely transcribed by r1 primer and r2 primer, respectively. Because of the inhibitory effect of ms²-modification to the reverse transcription, the amount of cDNA generated by r2 primer (Green lines) highly depends on the ms² levels in a given RNA sample. On the other hand, the cDNA generated by r1 primer (Blue lines) is independent of ms² level, and could be utilized as an internal control. The amount of each cDNA is quantified by a subsequent quantitative PCR (qPCR) using f1 and r1 primers.

1. For each tRNA species, mix DNase I-digested RNA with primer r1 or primer r2 on ice.
   

   Component	Volume (µl/reaction)
   	Sample (r1)	Sample (r2)
   DNase-digested RNA	2.5	2.5
   Primer r1 (10 µM)	1	―
   Primer r2 (10 µM)	―	1
   Water	3	3
   Total volume	6.5	6.5

   Note: It is recommended to prepare a master mixture of all reagents except RNA to reduce variability. When preparing a master mixture, it is usually desirable to prepare a slight excess to compensate for pipetting inaccuracies.
   
2. Denature RNA at 65 °C for 10 min and immediately put samples on ice for 1 min.
   
3. Add RT enzyme and buffer included in the Transcriptor First Strand cDNA Synthesis kit as follows on ice.
   

   Component	Volume (µl/reaction)
   	Sample (r1)	Sample (r2)
   Denatured RNA	6.5	6.5
   5x buffer	2	2
   RNase inhibitor	0.25	0.25
   dNTP Mix	1	1
   Enzyme	0.25	0.25
   Total volume	10	10

   Note: It is recommended to prepare a master mixture of all reagents except RNA to reduce variability. When preparing a master mixture, it is usually desirable to prepare a slight excess to compensate for pipetting inaccuracies.
   
4. Perform reverse transcription according to the following program.
   

   Time and Temperature
   Reaction	Inactivation	Hold
   55 °C for 30 min	85 °C for 5 min	4 °C

   
   
5. Samples are ready for quantitative PCR. Otherwise, samples can be stored at -20 °C.

Part III. Quantitative PCR

1. Mix reagents as follows. In case other SYBR green-based PCR kits are used, follow the manufacturer' instructions as recommended.
   

   Component	Volume (µl/reaction)
   	Sample (r1)	Sample (r2)
   SYBR Premix Ex Taq	10	10
   Primer f1 (10 µM)	0.4	0.4
   Primer r1 (10 µM)	0.4	0.4
   ROX reference dye	0.4	0.4
   cDNA	2	2
   Water	6.8	6.8
   Total volume	20	20

   Note: It is recommended to prepare a master mixture of all reagents except cDNA to reduce variability. When preparing a master mixture, it is usually desirable to prepare a slight excess to compensate for pipetting inaccuracies.
   
2. Set the program as follows and run real-time PCR. Add dissociation stage if required.
   

   Time and temperature
   First step	PCR (40 cycles)
   Heat-activation	Denaturing	Annealing/Elongation
   Hold	Cycle
   95 °C for 30 sec	95 °C for 5 sec	60 °C for 31 sec

   
   

Part IV. Data analysis
Analyze data from Samples (r1) and (r2) to obtain Ct values. The results obtained Samples (r1) and (r2) represents the total tRNA level and ms²-modification level in individual tRNA, respectively.
The normalized modification level in any given RNA sample is calculated as dCt = Ct (r2) – Ct (r1). Because the dCt value precisely reflects the modification level (Xie et al., 2013), the dCt value could be directly used for comparison of modification levels between multiple samples (see below for the representative data).

Representative data

Figure 2. Measurement of ms² level of mt-tRNA^Tyr in wild-type mouse heart tissue. Total RNA was purified from mouse heart tissue using TRIzol followed by 2-propanol precipitation. RNA sample was adjusted to 100 ng/µl and subjected to analysis as described above. The representative amplification curves using r1 primer (green) and r2 primer (blue) are shown. Ct (r2) = 14.8, Ct (r1) = 10.7. Modification level (dCt) in wild-type mouse heart = 14.8-10.7 = 4.1.


Figure 3. Measurement of ms² level of mt-tRNA^Tyr in heart tissue of Cdk5rap1 KO mouse. Total RNA was purified from heart tissue of Cdk5rap1 KO mouse that does not contain ms² modification. RNA sample was adjusted to 100 ng/µl and subjected to analysis as described above. The representative amplification curves using r1 primer (red) and r2 primer (blue) are shown. Ct (r2) = 10.4, Ct (r1) = 10.4. Modification level (dCt) in Cdk5rap1 KO mouse heart = 10.4-10.4 = 0.


Figure 4. Comparison of the modification levels of mt-tRNA^Tyr in heart tissues of wild-type (WT) and Cdk5rap1 KO mouse

Acknowledgments

The concept of this protocol was adapted from our previous study, in which the quantitative PCR was used to measure ms² modification in cytosolic tRNA^Lys(UUU) (Xie et al., 2013). We have successfully used this protocol to measure ms² modification mitochondrial tRNA in our recent study (Wei et al., 2015). This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References

1. Wei, F. Y., Zhou, B., Suzuki, T., Miyata, K., Ujihara, Y., Horiguchi, H., Takahashi, N., Xie, P., Michiue, H., Fujimura, A., Kaitsuka, T., Matsui, H., Koga, Y., Mohri, S., Suzuki, T., Oike, Y. and Tomizawa, K. (2015). Cdk5rap1-mediated 2-methylthio modification of mitochondrial tRNAs governs protein translation and contributes to myopathy in mice and humans. Cell Metab 21(3): 428-442.
   
2. Xie, P., Wei, F. Y., Hirata, S., Kaitsuka, T., Suzuki, T., Suzuki, T. and Tomizawa, K. (2013). Quantitative PCR measurement of tRNA 2-methylthio modification for assessing type 2 diabetes risk. Clin Chem 59(11): 1604-1612.