Comparison of two methods for RNA extraction from the nucleus pulposus of intervertebral discs.
RNA extraction from the nucleus pulposus of intervertebral discs has been extensively used in orthopedic studies. We compared two methods for extracting RNA from the nucleus pulposus: liquid nitrogen grinding and enzyme digestion. The RNA was detected by agarose gel electrophoresis, and the purity was evaluated by absorbance ratio using a spectrophotometer. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR). Thirty human lumbar intervertebral discs were used in this study. The liquid nitrogen-grinding method was used for RNA extraction from 15 samples, and the mean RNA concentration was 491.04 ± 44.16 ng/mL. The enzyme digestion method was used on 15 samples, and the mean RNA concentration was 898.42 ± 38.64 ng/mL. The statistical analysis revealed that there was a significant difference in concentration between the different methods. Apparent 28S, 18S, and 5S bands were detectable in RNA extracted using the enzyme digestion method, whereas no 28S or 18S bands were detected in RNA extracted using the liquid nitrogen-grinding method. The GAPDH band was visible, and no non-specific band was detected in the RT-PCR assay by the enzyme digestion method. Therefore, the enzyme digestion method is an efficient and easy method for RNA extraction from the nucleus pulposus of intervertebral discs for further intervertebral disc degeneration-related studies.
Department of Orthopaedic Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
M F
MF
Gan
Department of Orthopaedic Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
H L
HL
Yang
Department of Orthopaedic Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
J L
JL
Qian
Department of Orthopaedic Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
C S
CS
Wu
Department of Orthopaedic Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
C X
CX
Yuan
Department of Orthopaedic Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
X F
XF
Li
Department of Orthopaedic Surgery, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.
J
J
Zou
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis; D006801:Humans; D007403:Intervertebral Disc / Q000378:metabolism*; D055959:Intervertebral Disc Degeneration / Q000235:genetics*; D000070614:Nucleus Pulposus / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification*
D012313:RNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases