Difference between revisions of "Team:Uppsala/Transcriptomics/cDNA Conversion"

 
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<h2 id="Exp">Experiment</h2>
 
<h2 id="Exp">Experiment</h2>
  
     <p>The aim of this experiment was the conversion of RNA to cDNA through reverse transcription primed by oligo-dT. The process consists of 3 major steps - complementary DNA strand synthesis, RNA digestion and synthesis of second strand (IDT, 2018). The steps we conducted are described below:</p><br>
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     <p>The aim of this experiment was the conversion of RNA to cDNA through reverse transcription primed by oligo-dT. The process consists of 3 major steps - complementary DNA strand synthesis, RNA digestion and synthesis of second strand [1]. The steps we conducted are described below:</p><br>
  
 
     <h3>Synthesis of Complementary DNA Strand</h3>
 
     <h3>Synthesis of Complementary DNA Strand</h3>
<p>Due to previous polyA addition to 3´OH, all RNA molecules have similar sequence at end which only differs in number of added adenine bases. This allows using polyT primers (Oxford Nanopore) to anneal to RNA template and reverse transcriptase (SuperScript IV, ThermoFisher) can initiate the transcription. <br>
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<p>Due to previous polyA addition to 3'OH, all RNA molecules have similar sequence at 3' end which only differs in number of added adenine bases. This allows us to use polyT primers (Oxford Nanopore) to anneal to RNA template and reverse transcriptase (SuperScript IV, ThermoFisher) can initiate the transcription. <br>
  
 
A second, so-called strand switching primer is added to the reaction. This compensates for under-representations of 5´ends in cDNA by introducing an additional template and therefore protecting the terminal base pairs. Terminal transferase activity of the RT adds a number of deoxycytidine bases. The SSP primer is complementary to these bases and acts as an extended template for the RT, not only protecting the terminal bases, but also allowing to introduce sequence of choice into the newly synthesized first strand.</p> <br>
 
A second, so-called strand switching primer is added to the reaction. This compensates for under-representations of 5´ends in cDNA by introducing an additional template and therefore protecting the terminal base pairs. Terminal transferase activity of the RT adds a number of deoxycytidine bases. The SSP primer is complementary to these bases and acts as an extended template for the RT, not only protecting the terminal bases, but also allowing to introduce sequence of choice into the newly synthesized first strand.</p> <br>
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   <h3> RNA Contamination</h3>
 
   <h3> RNA Contamination</h3>
<p>cDNA which was synthesized during this experiment was used to prepare sequencing library. Due to the suboptimal sequencing performance as described <a href="https://2018.igem.org/Team:Uppsala/Transcriptomics/Sequencing"> here</a>, we began to investigate among other factors the quality of the input cDNA. After extensive troubleshooting it was determined that RNA was still present in the cDNA samples despite digestion and cleaning steps. In several samples very high amount of RNA has been found, often corresponding to the input quantity. RNA content was measured using Qubit HS RNA Kit specific to RNA.  Unfortunately, efficient way of removing RNA has not been found during the course of the project, which is the main reason for poor sequencing results.</p></br>
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<p>cDNA which was synthesized during this experiment was used to prepare the sequencing library. Due to the suboptimal sequencing performance as described <a href="https://2018.igem.org/Team:Uppsala/Transcriptomics/Sequencing"> here</a>, we began to investigate among other factors the quality of the input cDNA. After extensive troubleshooting it was determined that RNA was still present in the cDNA samples despite digestion and cleaning steps. In several samples very high amount of RNA has been found, often corresponding to the input quantity. RNA content was measured using Qubit HS RNA Kit specific to RNA.  Unfortunately, an efficient way of removing RNA has not been found during the course of the project, which is the main reason for poor sequencing results.</p></br>
  
 
<p>The following section describes the various troubleshooting approaches to investigating the RNA contamination.</p>
 
<p>The following section describes the various troubleshooting approaches to investigating the RNA contamination.</p>
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<h4>Hypothesis:</h4>  
 
<h4>Hypothesis:</h4>  
<p>The measured RNA in the sample could be caused due to lack of specificity of Qubit dye eg. RNA dye actually has affinity to DNA and therefore shows RNA in our samples.</p>
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<p>The measured RNA in the sample could be due to lack of specificity of Qubit dye eg. RNA dye actually has affinity to DNA and therefore shows RNA in our samples.</p>
  
 
<h4>Experiment:</h4>  
 
<h4>Experiment:</h4>  
<p>Samples containing only RNA or DNA in concentration of 10 ng/µl were prepared in triplicate and each measured with two different Qubit kits (RNA HS Kit, DNA HS Kit, Thermo Fisher). </p>
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<p>Samples containing only RNA or DNA in concentration of 10 ng/µl were prepared in triplicate and each sample was measured with two different Qubit kits (RNA HS Kit, DNA HS Kit, Thermo Fisher). </p>
  
 
<h4>Results:</h4>  
 
<h4>Results:</h4>  
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<h4>Conclusion</h4>
 
<h4>Conclusion</h4>
<p>The experiment only investigated interactions between dsDNA and ssRNA, which are not supposed to introduce any bias into the measurement as per manufacturer's information (ThermoFischer, 2018). The remaining question is how would a RNA:DNA hybrid be treated by the dye. It is possible that RNA was not properly digested and therefore remains in the hybrid form. A hybrid could hypothetically be detected by both RNA and DNA specific kit. Unfortunately no proven hybrid sample was available and therefore this hypothesis could not be tested. <br><br>
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<p>The experiment only investigated interactions between dsDNA and ssRNA, which are not supposed to introduce any bias into the measurement as per manufacturer's information [2]. The remaining question is how would a RNA:DNA hybrid be treated by the dye. It is possible that RNA was not properly digested and therefore remains in the hybrid form. A hybrid could hypothetically be detected by both RNA and DNA specific kit. Unfortunately no proven hybrid sample was available and therefore this hypothesis could not be tested. <br><br>
  
It was concluded that having DNA - RNA mixture does not influence the measurement in a significant way. RNA amount is moderately decreased after addition of DNA into the sample, which would not explain the presence of RNA in cDNA samples. What remains to be investigated is how would a RNA:DNA hybrid influence the measurement. We therefore conclude that the RNA measurement in our samples is accurate and there is RNA present, either as a ssRNA of RNA:DNA hybrid. </p><br><br>
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It was concluded that having DNA - RNA mixture does not influence the measurement in a significant way. RNA amount is moderately decreased after addition of DNA into the sample, which would not explain the presence of RNA in cDNA samples. What remains to be investigated is how a RNA:DNA hybrid would influence the measurement. We therefore conclude that the RNA measurement in our samples is accurate and there is RNA present, either as a ssRNA or RNA:DNA hybrid. </p><br><br>
  
 
<h3>Is RNA Template Properly Digested? Is It Carried Over with the AMPure XP Beads?</h3>
 
<h3>Is RNA Template Properly Digested? Is It Carried Over with the AMPure XP Beads?</h3>
  
 
<h4>Hypothesis:</h4>  
 
<h4>Hypothesis:</h4>  
<p>RNA is found in the final cDNA product as it is not digested properly. The Rnase Cocktail contains RNAse A and T1, which are only supposed to introduce nicks into the RNA. Provided that LongAmp Taq polymerase does not degrade these fragment and that the purification beads (AMPure XP, Agencourt) do have affinity to both RNA and DNA, the RNA would be found in the sample. </p>
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<p>RNA is found in the final cDNA product as it is not digested properly. The RNase Cocktail contains RNAse A and T1, which are only supposed to introduce nicks into the RNA. Provided that LongAmp Taq polymerase does not degrade these fragment and that the purification beads (AMPure XP, Agencourt) do have affinity to both RNA and DNA, the RNA would be found in the sample. </p>
  
 
<h4>Experiment:</h4>  
 
<h4>Experiment:</h4>  
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<h4>Results:</h4>  
 
<h4>Results:</h4>  
<p>Overall, it was seen that subsequent digestion of RNA contaminated samples was successful regardless of used concentration as shown below. Digestion of RNA ladder using RNase H and/or Rnase Cocktail shows complete digestion for RNase Cocktail and RNase H seems not to be working. The beads do carry over most of the RNA including rather small fragments (200 bp). The detailed results of each experiments are shown below:<br></p>
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<p>Overall, it was seen that subsequent digestion of RNA contaminated samples was successful regardless of used concentration as shown below. Digestion of RNA ladder using RNase H and/or RNase Cocktail shows complete digestion for RNase Cocktail while RNase H seems to not be working. The beads do carry over most of the RNA including rather small fragments (200 bp). The detailed results of each experiments are shown below:<br></p>
 
<br>
 
<br>
 
<p><b>1.</b></p>
 
<p><b>1.</b></p>
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<p>According to measurement with Qubit RNA HS, all samples regardless of treatment show all RNA being degraded including sample, where treatment is identical to the one used in the actual experiment. </p><br>
 
<p>According to measurement with Qubit RNA HS, all samples regardless of treatment show all RNA being degraded including sample, where treatment is identical to the one used in the actual experiment. </p><br>
  
<p><b>2.</b> The gel below in Figure 2 shows results of digestion of RNA ladder with the available RNases, Rnase H (line 5 and 6) and RNase Cocktail (line 7 and 8) or both (line 8 and 9).<br><br>
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<p><b>2.</b> The gel below in Figure 2 shows results of the digestion of RNA ladder with the available RNases, RNase H (line 5 and 6) and RNase Cocktail (line 7 and 8) or both (line 8 and 9).<br><br>
  
From this experiment we can conclude that the buffer has no effect on digestion since sample in water and in reaction buffer appear the same. It can further be said that RNase Cocktail efficiently degrades the ladder, which is composed of ssRNA. RNAseH seems to not digest the ladder at all. <br><br>
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From this experiment we can conclude that the buffer has no effect on digestion since sample in water and in reaction buffer appear the same. It can further be said that RNase Cocktail efficiently degrades the ladder, which is composed of ssRNA. RNase H seems to not digest the ladder at all. <br><br>
  
This corresponds with described ability of RNAseH to preferentially digest RNA:DNA hybrids.</p>
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This corresponds with described ability of RNase H to preferentially digest RNA:DNA hybrids.</p>
 
      
 
      
 
     </div>
 
     </div>
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<p>During the cDNA synthesis it was possible to achieve sufficiently high yields, usually exceeding double of the input mRNA amount. This cDNA was used to prepare sequencing libraries. As was shown later, it unfortunately contained undigested RNA, which significantly decrease the quality of sequencing results. We have therefore spend significant amount of time searching for the source of RNA contamination as reported in this section. <br><br>
 
<p>During the cDNA synthesis it was possible to achieve sufficiently high yields, usually exceeding double of the input mRNA amount. This cDNA was used to prepare sequencing libraries. As was shown later, it unfortunately contained undigested RNA, which significantly decrease the quality of sequencing results. We have therefore spend significant amount of time searching for the source of RNA contamination as reported in this section. <br><br>
  
The experiments above show very contradicting results. In section i) it can be seen that treatment of cDNA with RNAse Cocktail after synthesis resulted in complete clearance of RNA from the sample. RNase Cocktail has also been shown to digest RNA ladder as visualized on the gel in Figure 1.
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The experiments above show very contradicting results. In section i) it can be seen that treatment of cDNA with RNase Cocktail after synthesis resulted in complete clearance of RNA from the sample. RNase Cocktail has also been shown to digest RNA ladder as visualized on the gel in Figure 1.
  
 
The same enzyme has always been used during the cDNA synthesis procedure and it remains unclear why does it efficiently digest RNA after synthesis or RNA ladder and would not work during the actual synthesis. <br><br>
 
The same enzyme has always been used during the cDNA synthesis procedure and it remains unclear why does it efficiently digest RNA after synthesis or RNA ladder and would not work during the actual synthesis. <br><br>
  
One hypothesis that we considered was the presence of RNA:DNA hybrids, which would decrease efficiency of RNAse Cocktail, which is more efficient in digesting ssDNA. RNAse H was therefore added to address this issue but clearance of RNA from samples did not significantly increase. Even more confusingly, digestion did work in some of the samples despite the content / treatment being identical. <br><br>
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One hypothesis that we considered was the presence of RNA:DNA hybrids, which would decrease efficiency of RNase Cocktail, which is more efficient in digesting ssDNA. RNase H was therefore added to address this issue but clearance of RNA from samples did not significantly increase. Even more confusingly, digestion did work in some of the samples despite the content/treatment being identical. <br><br>
  
 
<h2 id="Conc">Conclusion</h2>
 
<h2 id="Conc">Conclusion</h2>
<p>We have managed to successfully synthesize complementary DNA to our mRNA samples, which unfortunately did contain undigested RNA. A protocol needs to be developed that assures all of the RNA has been removed from the sample prior to the preparation of the library. Moreover, additional troubleshooting needs to be performed to determine why is the digestion not efficient. </p>
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<p>We have managed to successfully synthesize complementary DNA to our mRNA samples, which unfortunately did contain undigested RNA. A protocol needs to be developed that assures all of the RNA has been removed from the sample prior to the preparation of the library. Moreover, additional troubleshooting needs to be performed to determine why the digestion is not efficient. </p>
  
  
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<p><b>[1]</b> IDT, Use of template switching oligos (TS oligos, TSOs) for efficient cDNA library construction, [online], 2018  
 
<p><b>[1]</b> IDT, Use of template switching oligos (TS oligos, TSOs) for efficient cDNA library construction, [online], 2018  
  <a href="https://eu.idtdna.com/pages/education/decoded/article/use-of-template-switching-oligos-(ts-oligos-tsos)-for-efficient-cdna-library-construction">https://eu.idtdna.com/pages/education/decoded/article/use-of-template-switching-oligos-(ts-oligos-tsos)-for-efficient-cdna-library-construction</a> </p><br>
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  <a href="https://eu.idtdna.com/pages/education/decoded/article/use-of-template-switching-oligos-(ts-oligos-tsos)-for-efficient-cdna-library-construction">https://eu.idtdna.com/pages/education/decoded/article/use-of-template-switching-oligos-(ts-oligos-tsos)-for-efficient-cdna-library-construction</a> </p>
  
<p><b>[1]</b> Thermo Fisher Scientific Inc, User Guide: Qubit RNA HS Assay Kits, [online], 2015  
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<p><b>[2]</b> Thermo Fisher Scientific Inc, User Guide: Qubit RNA HS Assay Kits, [online], 2015  
 
  <a href="https://www.thermofisher.com/order/catalog/product/Q32852">https://www.thermofisher.com/order/catalog/product/Q32852</a> </p><br>
 
  <a href="https://www.thermofisher.com/order/catalog/product/Q32852">https://www.thermofisher.com/order/catalog/product/Q32852</a> </p><br>
 
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Latest revision as of 16:03, 3 December 2018