Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

An efficient and cost-effective method for purification of small sized DNAs and RNAs from human urine.

Author(s): Zainabadi K, Dhayabaran V, Moideen K, Krishnaswamy P

Publication: PLoS One, 2019, Vol. 14, Page e0210813

PubMed ID: 30721243 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of lysis buffer composition, ratio of urine to lysis buffer, addition of BSA or casein to the urine, filtration plate type, and wash solution components on the recovery of spiked-in DNA fragments of different sizes from urine. Excess first-morning urine from an unspecified number of healthy males were pooled and spiked with 100 bp DNA ladder. Lysis buffer containing guanidine thiocyanate (3 M, 4 M, or 6 M) or 3M guanidine hydrochloride, 20-33% isopropanol or ethanol, 0-1% 2-mercaptoethanol in addition to 20 mM TrisHCl, 50 mM EDTA, and 4% Triton-X100 at pH 6.0 or 6.5 was mixed with urine at a ratio of 1:1 or 2:1. The mixture was placed into filter plates (Nunc, Omega EZ, Econospin, or Pureplate), centrifuged at 2250 x g for 1 min, and washed in a solution containing lysis buffer without 2-mercaptoethanol. The plate was centrifuged again at 2250 x g for 1 min; washed in 25% ethanol, 25% isopropanol, 10 mM NaCl, and 10 mM Tris pH 7.4; and centrifuged again at 2250 x g for 2 min. The plate was then dried and DNA was eluted with TE buffer (pH 8.0). DNA ladder was quantified by electrophoresis. Extracted DNA was reverse-transcribed and total DNA isolated was quantified by real-time PCR amplification of actin.

   Summary of Findings:

   Recovery of DNA larger than 100 bp was not affected by the pH of the lysis buffer, but recovery of the 100 bp band was only possible when the pH of the lysis buffer was ≤6.5 or contained ethanol (15 or 20%). The average CT for actin was lowest when the lysis solution contained 3 M guanidine thiocyanate (rather than 4 M or 6 M guanidine thiocyanate or 3 M guanidine hydrochloride), 33% isopropanol (rather than 20% isopropanol or 20 or 33% ethanol), and 0.5% 2-mercaptoethanol (rather than 0%, 0.1%, 0.25%, 0.33%, or 1%) and was used at a ratio of 1:1 (rather than 2:1). There was no effect of using a lysis buffer with a pH of 6.0 versus 6.5 on the average CT value of actin. Interestingly, the addition of BSA to the lysis buffer had no effect or increased the average CT, but adding BSA to the urine directly resulted in a lower average actin CT with the lowest average CT achieved with the addition of 1 or 3.3 mg/ml (compared with 0, 0.1, 0.33, or 10 mg/mL) to the urine depending on the plate used. The addition of isopropanol to the lysis buffer only affected the average CT value when a reverse-transcription step was included, indicating it enhanced the co-purification of RNA. In conjunction with the optimized lysis buffer, the lowest average CT value of actin for combined lysis urine volumes of ≤800 µL was when Nunc 96-well filter plates were used (compared with Omega EZ, Econospin, or Pureplate). Interestingly, the components of the wash solution (0% or 0.5% 2-mercaptoethanol, 0.1 mg/mL or 1 mg/mL BSA, or 0.1 mg/mL or 1 mg/mL α-casein) had little to no effect on the average CT value of actin (all changes were within SD).

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Lysis buffer containing 3 M guanidine thiocyanate and 33% isopropanol Lysis buffer containing 4 M guanidine thiocyanate and 33% isopropanol Lysis buffer containing 6 M guanidine thiocyanate and 33% isopropanol Lysis buffer containing 3 M guanidine thiocyanate and 20% isopropanol Lysis buffer containing 4 M guanidine thiocyanate and 20% isopropanol Lysis buffer containing 6 M guanidine thiocyanate and 20% isopropanol Lysis to urine 1:1 Lysis to urine 2:1 Lysis buffer pH 6.5 Lysis buffer pH 6.0 Lysis buffer pH 7.5 Lysis buffer with 0% 2-mercaptoethanol Lysis buffer with 0.1% 2-mercaptoethanol Lysis buffer with 0.33% 2-mercaptoethanol Lysis buffer with 1% 2-mercaptoethanol Lysis buffer with 0.25% 2-mercaptoethanol Lysis buffer with 0.5% 2-mercaptoethanol
   Analyte Extraction and Purification	Washing	Containing 0% 2-mercaptoethanol Containing 0.5% 2-mercaptoethanol Containing 0.1 mg/mL BSA Containing 1 mg/mL BSA Containing 0.1 mg/ mL α-casein Containing 1 mg/ mL α-casein
   Biospecimen Aliquots and Components	Biospecimen components	0 mg/mL BSA added to urine 0.1 mg/mL BSA added to urine 0.33 mg/mL BSA added to urine 1 mg/mL BSA added to urine 3.3 mg/mL BSA added to urine 10 mg/mL BSA added to urine
   Analyte Extraction and Purification	Analyte purification	Nunc plate Omega EZ Econospin Pureplate

   View more

2. Study Purpose

   This study investigated the effects of urine volume, volume of silica, and the use of size exclusion plates on the isolation of DNA from urine. Excess first-morning urine from an unspecified number of healthy males were pooled. Lysis buffer containing 3 M guanidine thiocyanate, 33% isopropanol, 0.5% 2-mercaptoethanol, 20 mMTrisHCl, 50 mM EDTA, and 4% Triton-X100 at pH 6.0 was mixed with urine at a ratio of 1:1. Silica (125-1000 µL) was added to the lysis solution, centrifuged at 3000 rpm, and resuspended in a lysis buffer without 2-mercaptoethanol. The mixture was applied to a size exclusion plate or left in the tubes, centrifuged at 2250 x g for 2 min, and washed in lysis buffer. The plate was centrifuged again at 2250 x g for 2 min; washed in 25% ethanol, 25% isopropanol, 10 mM NaCl, and 10 mMTris pH 7.4; and centrifuged again at 2250 x g for 2 min. The plate was then dried and DNA was eluted with TE buffer (pH 8.0). Extracted DNA was reverse-transcribed and total DNA isolated was quantified by real-time PCR amplification of actin.

   Summary of Findings:

   For larger urine volumes (>800 µL), the lowest average CT value of actin was achieved through the use of 250 µL silica (as opposed to 125, 500, or 1000 µL) and Pall GHP 0.45 µm size-exclusion plates (rather than a Pall Supor 1.2 µm size -exclusion plate or use of a tube). As the input volume was adjusted from 250 µL to 2.5 mL and 25 mL, the average Ct values decreased by 2.5 and 5.2, respectively; indicating that the method can be adapted for low abundance targets by increasing urine volume.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot size/volume	250 µL 2.5 mL 25 mL
   Analyte Extraction and Purification	Analyte purification	125 µL silica 250 µL silica 500 µL silica 1000 µL silica Pall GHP 0.45µM Pall Supor 1.2 µm Tube

   View more

3. Study Purpose

   This study compared the amount of the DNA in cellular versus non-cellular components in urine specimens by centrifugation at two speeds and investigated the size of the isolated DNA using real-time PCR of different fragment sizes of actin.  Pooled urine from an unspecified number of healthy males was centrifuged at 7500 rpm (5283 x g) or 15000 rpm (21130 x g) for 10 min. The resultant supernatant and pellets were mixed with lysis solution (3 M guanidine thiocyanate, 33% isopropanol, 0.5% 2-mercaptoethanol, 20 mM Tris HCl, 50 mM EDTA, and 4% Triton-X100 at pH 6.0). The mixture was placed into Nunc filter plates, centrifuged at 2250 x g for 1 min, and washed in a solution containing lysis buffer without 2-mercaptoethanol. The plate was centrifuged again at 2250 x g for 1 min; washed in 25% ethanol, 25% isopropanol, 10 mM NaCl, and 10 mM Tris pH 7.4; and centrifuged again at 2250 x g for 2 min. The plate was then dried and DNA was eluted with TE buffer (pH 8.0). Extracted DNA was reverse transcribed and total DNA isolated was quantified by real-time PCR amplification of actin of different sizes (60, 92, 180, 241, and 180 bp).

   Summary of Findings:

   The highest yield of DNA was amplified from whole urine with lower but similar amounts of DNA present in the pellets and supernatant (both at 7500 rpm and 15000 rpm). The authors state that this shows cellular and non-cellular DNA are equally represented in urine. The supernatant and 7500 rpm pellets had comparable amplification of the 60 bp, 92 bp, and 180 bp fragments in the pellets and supernatant, but the supernatant had considerably less amplification of the 241 bp and 480 bp fragments. The authors state this confirms that it is important to use amplicons of 180 bp or less when analyzing cell-free DNA.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qPCR Specific	Length of gene fragment	60 bp 92 bp 180 bp 241 bp 480 bp
   Biospecimen Aliquots and Components	Centrifugation	Multiple speeds compared
   Biospecimen Aliquots and Components	Biospecimen components	Supernatant Pellet Whole urine

   View more

4. Study Purpose

   This study investigated the effects of storing urine for 1 week at room temperature, 4˚C, -20˚C, or  -80˚C on cell-free DNA levels. The effects of preservation with EDTA with or without sodium azide, lysis buffer containing 3 M guanidine thiocyanate and 33% isopropanol, or the Qiagen RLT buffer with isopropanol on the stability of cell free DNA in urine stored at room temperature was also investigated. Pooled urine from an unspecified number of healthy males was stored for 1 week at room temperature, 4˚C, -20˚C, or -80˚C or preserved with EDTA with or without sodium azide, lysis buffer containing 3 M guanidine thiocyanate and 33% isopropanol, or Qiagen RLT buffer with isopropanol and stored at room temperature for 1 or 2 weeks (lysis buffer and RLT only). After storage, urine was mixed with lysis solution (3 M guanidine thiocyanate, 33% isopropanol, 0.5% 2-mercaptoethanol, 20 mM Tris HCl, 50 mM EDTA, and 4% Triton-X100 at pH 6.0). The mixture was placed into Nunc filter plates, centrifuged at 2250 x g for 1 min, and washed in a solution containing lysis buffer without 2-mercaptoethanol. The plate was centrifuged again at 2250 x g for 1 min; washed in 25% ethanol, 25% isopropanol, 10 mM NaCl, and 10 mM Tris pH 7.4; and centrifuged again at 2250 x g for 2 min. The plate was then dried and DNA was eluted with TE buffer (pH 8.0). Extracted DNA was reverse-transcribed and total DNA isolated was quantified by real-time PCR amplification of actin.

   Summary of Findings:

   As expected, average CT values increased when urine was stored for 1 week at room temperature. This increase was partially attenuated by the addition of EDTA with or without sodium azide and by refrigeration; however, the addition of the lysis buffer containing 3 M guanidine thiocyanate and 33% isopropanol resulted in lower CT values than even urine frozen at -20 or -80˚C. Further study showed that 3 M guanidine thiocyanate and 33% isopropanol provided comparable stability to the Qiagen RLT buffer with isopropanol for up to 2 weeks.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Other Preservative
   • Frozen
   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	EDTA Sodium azide Refrigeration Frozen Guanidine thiocyanate Qiagen RLT buffer
   Storage	Storage temperature	-20˚C -80˚C Room temperature
   Storage	Storage duration	1 week 2 weeks

   View more