contain a 5
0 (tri-)phosphate group, such as home-transcribed
RNA markers, the CIP treatment is needed.
7. To avoid obtaining lower phase, you are suggested to use a
200 μL pipette to decant upper phase several times.
8. Do not over-dry RNA pellet, which might cause difficulties in
RNA dissolvement and RNA degradation.
9. The absorbance ratio of RNA with good quality at 260 and
280 nm (A260/280) should be between 1.8 and 2.0. Gel
electrophoresis result should display a 28S/18S ratio as 2/1.
10. After this step, one can use fastQC (Table 3) to check the
distribution of reads in different lengths. Usually reads amount
of 24 nt should be higher or even about twice of that of 21 nt
except some tissues with actively dividing and developing cells
[39]. If the samples are from materials including vegetative
organs or inflorescence buts, 24 nt reads amount is similar or
even lower than 21 nt reads amount, and the RNA might have
a certain level of degradation.
11. Make sure that the chromosome names in the index file used
for bowtie alignment are consistent with those in the gff3 file.
Acknowledgments
The work in the X. Zhang’s laboratory was supported by the NIH
grant R01GM132401. D.S. was supported by the China Scholar
Council Fellowship.
References
1. Ma Z, Zhang X (2018) Actions of plant argonautes: predictable or unpredictable? Curr
Opin Plant Biol 45:59–67
2. Bartel DP (2004) Micrornas: genomics, biogenesis, mechanism, and function. Cell
116:281–297
3. Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of
small rna. Nat Protoc 3:1077
4. Kroh EM, Parkin RK, Mitchell PS et al (2010)
Analysis of circulating microrna biomarkers in
plasma and serum using quantitative reverse
transcription-pcr
(qrt-pcr).
Methods
50:298–301
5. Chen C, Ridzon DA, Broomer AJ et al (2005)
Real-time quantification of micrornas by
stem–loop rt–pcr. Nucleic Acids Res 33:
e179–e179
6. Git A, Dvinge H, Salmon-Divon M et al
(2010) Systematic comparison of microarray
profiling, real-time pcr, and next-generation
sequencing technologies for measuring differential microrna expression. RNA 16:991–1006
7. Hafner M, Landgraf P, Ludwig J et al (2008)
Identification of micrornas and other small regulatory rnas using cdna library sequencing.
Methods 44:3–12
8. Wang H, Zhang X, Liu J et al (2011) Deep
sequencing of small rnas specifically associated
with arabidopsis ago1 and ago4 uncovers new
ago functions. Plant J 67:292–304
9. Williams Z, Ben-Dov IZ, Elias R et al (2013)
Comprehensive profiling of circulating microrna via small rna sequencing of cdna libraries
reveals biomarker potential and limitations.
Proc Natl Acad Sci U S A 110:4255–4260
252
Di Sun et al.
0 (tri-)phosphate group, such as home-transcribed
RNA markers, the CIP treatment is needed.
7. To avoid obtaining lower phase, you are suggested to use a
200 μL pipette to decant upper phase several times.
8. Do not over-dry RNA pellet, which might cause difficulties in
RNA dissolvement and RNA degradation.
9. The absorbance ratio of RNA with good quality at 260 and
280 nm (A260/280) should be between 1.8 and 2.0. Gel
electrophoresis result should display a 28S/18S ratio as 2/1.
10. After this step, one can use fastQC (Table 3) to check the
distribution of reads in different lengths. Usually reads amount
of 24 nt should be higher or even about twice of that of 21 nt
except some tissues with actively dividing and developing cells
[39]. If the samples are from materials including vegetative
organs or inflorescence buts, 24 nt reads amount is similar or
even lower than 21 nt reads amount, and the RNA might have
a certain level of degradation.
11. Make sure that the chromosome names in the index file used
for bowtie alignment are consistent with those in the gff3 file.
Acknowledgments
The work in the X. Zhang’s laboratory was supported by the NIH
grant R01GM132401. D.S. was supported by the China Scholar
Council Fellowship.
References
1. Ma Z, Zhang X (2018) Actions of plant argonautes: predictable or unpredictable? Curr
Opin Plant Biol 45:59–67
2. Bartel DP (2004) Micrornas: genomics, biogenesis, mechanism, and function. Cell
116:281–297
3. Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of
small rna. Nat Protoc 3:1077
4. Kroh EM, Parkin RK, Mitchell PS et al (2010)
Analysis of circulating microrna biomarkers in
plasma and serum using quantitative reverse
transcription-pcr
(qrt-pcr).
Methods
50:298–301
5. Chen C, Ridzon DA, Broomer AJ et al (2005)
Real-time quantification of micrornas by
stem–loop rt–pcr. Nucleic Acids Res 33:
e179–e179
6. Git A, Dvinge H, Salmon-Divon M et al
(2010) Systematic comparison of microarray
profiling, real-time pcr, and next-generation
sequencing technologies for measuring differential microrna expression. RNA 16:991–1006
7. Hafner M, Landgraf P, Ludwig J et al (2008)
Identification of micrornas and other small regulatory rnas using cdna library sequencing.
Methods 44:3–12
8. Wang H, Zhang X, Liu J et al (2011) Deep
sequencing of small rnas specifically associated
with arabidopsis ago1 and ago4 uncovers new
ago functions. Plant J 67:292–304
9. Williams Z, Ben-Dov IZ, Elias R et al (2013)
Comprehensive profiling of circulating microrna via small rna sequencing of cdna libraries
reveals biomarker potential and limitations.
Proc Natl Acad Sci U S A 110:4255–4260
252
Di Sun et al.