BACKGROUND: Assessment of mRNA levels has the potential to predict renal outcome. The objective of this study was to optimize several steps in the protocol for obtaining cDNA from routine clinical kidney biopsy material. METHODS: RNA integrity was compared between different methods for extraction of RNA, synthesis of cDNA, and storage of renal tissue. Hereby, RNAlater, an RNA-preserving compound, was tested for implementation in a protocol for renal biopsies that combines routine histology and RNA expression studies. Gel electrophoresis and real-time polymerase chain reactions (PCR) were outcome parameters for assessment of RNA integrity. RESULTS: The Trizol method rendered higher RNA yields from fresh renal tissue than the NP40 method and RNeasy spin columns. RNA yields were not affected when renal tissue was stored at -70 degrees C for up to 2 months in phosphate-buffered saline (PBS). cDNA levels obtained using avian myeloblastosis virus (AMV) reverse transcriptase (RT) were at least twice as high as those obtained with Sensiscript and Superscript RT. RNAlater maintained RNA integrity in whole renal cortex stored at 4 degrees C for up to 3 months. Dissection of small biopsies in RNAlater rendered similar RNA yields in comparison with dissection in PBS, but the yield of glomeruli from the cortices was 50% lower (P < 0.005). Integrity of RNAlater-treated tissue, evaluated by light microscopy and immunofluorescence, was diminished. CONCLUSION: This study shows optimization of several steps in the protocol for extraction and handling of RNA in renal cortical tissue. RNA extraction and cDNA synthesis can be optimized by the use of the Trizol method and AMV RT, respectively. RNAlater is beneficial for preserving RNA integrity in whole renal cortex during storage and processing, but is not suitable for implementation in routine diagnostic histologic stainings combined with RNA expression studies in dissected biopsy material.
BACKGROUND: Assessment of mRNA levels has the potential to predict renal outcome. The objective of this study was to optimize several steps in the protocol for obtaining cDNA from routine clinical kidney biopsy material. METHODS: RNA integrity was compared between different methods for extraction of RNA, synthesis of cDNA, and storage of renal tissue. Hereby, RNAlater, an RNA-preserving compound, was tested for implementation in a protocol for renal biopsies that combines routine histology and RNA expression studies. Gel electrophoresis and real-time polymerase chain reactions (PCR) were outcome parameters for assessment of RNA integrity. RESULTS: The Trizol method rendered higher RNA yields from fresh renal tissue than the NP40 method and RNeasy spin columns. RNA yields were not affected when renal tissue was stored at -70 degrees C for up to 2 months in phosphate-buffered saline (PBS). cDNA levels obtained using avian myeloblastosis virus (AMV) reverse transcriptase (RT) were at least twice as high as those obtained with Sensiscript and Superscript RT. RNAlater maintained RNA integrity in whole renal cortex stored at 4 degrees C for up to 3 months. Dissection of small biopsies in RNAlater rendered similar RNA yields in comparison with dissection in PBS, but the yield of glomeruli from the cortices was 50% lower (P < 0.005). Integrity of RNAlater-treated tissue, evaluated by light microscopy and immunofluorescence, was diminished. CONCLUSION: This study shows optimization of several steps in the protocol for extraction and handling of RNA in renal cortical tissue. RNA extraction and cDNA synthesis can be optimized by the use of the Trizol method and AMV RT, respectively. RNAlater is beneficial for preserving RNA integrity in whole renal cortex during storage and processing, but is not suitable for implementation in routine diagnostic histologic stainings combined with RNA expression studies in dissected biopsy material.
Authors: Kazumi Taguchi; Manint Usawachintachit; Shuzo Hamamoto; Rei Unno; David T Tzou; Benjamin A Sherer; Yongmei Wang; Atsushi Okada; Marshall L Stoller; Takahiro Yasui; Thomas Chi Journal: J Endourol Date: 2017-08-11 Impact factor: 2.942
Authors: Carlos P Vio; Mariana Quiroz-Munoz; Catherina A Cuevas; Carlos Cespedes; Nicholas R Ferreri Journal: Am J Physiol Renal Physiol Date: 2012-05-23
Authors: Gaby Hoffmann; Jooske Ijzer; Bas Brinkhof; Baukje A Schotanus; Ted S G A M van den Ingh; Louis C Penning; Jan Rothuizen Journal: Comp Hepatol Date: 2009-07-08